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Analysis of Nuclear Pore Complex Phosphorylation Sites using a MALDI-LTQ Orbitrap Mass Spectrometer 1 1 1 2 Justin Blethrow , Rosa Viner , Vlad Zabrouskov , and Joseph Glavy 1 Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134 2 Stevens Institute of Technology, Castle Point on Hudson, Hoboken NJ 07030-5991 Introduction The nuclear pore complex (NPC) were found to be specific or substantially enriched contains ~30 proteins and forms the mainl in mitotic proteins. Additional phosphopeptides passageway for nucleo-cytoplasmic were detected in proteins isolated using mab414, macromolecular traffic. In higher eukaryotes, the including several FG-repeat proteins and Nup358. NPC undergoes mitotic disassembly into The ongoing work is focused on expanding our subcomplexes, thought to be enacted largely via analysis for fuller coverage of these and other phosphorylation. We used affinity isolation and NPC components. high mass accuracy LC MALDI MS and MS/MS to identify phosphorylation sites in the NPC and to Innovative aspects quantify their relative mitotic and interphase • Analysis of MALDI-produced ions with an abundances by stable isotope labeling. orbital trap / ion trap hybrid mass spectrometer. Methods HeLa cells grown in the presence of • Antibody-based dissection of the Nuclear Pore labeled arginine or lysine were arrested in G1 and Complex. lysed. This was added to unlabeled lysate from synchronous mitotic cells previously released from References G1 arrest. Immunoprecipitations were performed 1: Glavy JS, et al.; Proc. Natl. Acad. Sci. – 104, using mab414 to isolate approximately ten FG- 3811-3816 (2007); Cell-cycle-dependent repeat and associated proteins, and an antibody to phosphorylation of the nuclear pore Nup107–160 Nup107, isolating the Nup107-160 subcomplex. subcomplex. Proteins were enzymatically digested, and peptides were separated by HPLC with automated fraction spotting. Alternatively, proteins were separated by PAGE, excised, and processed for MALDI. Spots were analyzed by MALDI-LTQ Orbitrap, using survey scans of 60k nominal resolution. Phosphopeptides were detected by Neutral Loss ion mapping in the LTQ and selected for MS3 analysis or HCD fragmentation in the Orbitrap. Results Immunoprecipitation with anti-Nup107 purified the Nup107-160 subcomplex. MS analysis identified nine proteins, similar to previous studies(1). Immunoprecipitation with mab414 purified seven NPC components (Nup358, 214,153, 88, 62, 58 and 54) and two karyopherin proteins (nucleo-cytoplasmic transporters). Full MS analysis using the Orbitrap consistently provided high mass accuracies (3 ppm RMS), which significantly increased the confidence of peptide identifications. The LC-MALDI analysis format allowed for fragmentation analysis of every mass value at every LC time point. This provided sensitive detection of phosphopeptides by monitoring for neutral loss of phosphate in MS2 3 (ion mapping). Subsequent manual MS analysis to localize the phosphorylation site was performed if the precursor was not previously selected automatically in the standard data dependent experiment. Numerous phosphorylation sites were observed in the Nup107-160 subcomplex. Consistent with previous results, several of these Analyses of Proteasome Complexes and Proteome Variations in Acute Myeloid Leukemia Cells 1 1 1 2 2 1 M. Matondo , M.P. Bousquet-Dubouch , S. Uttenweiler-Joseph , B. Payrastre , S. Manenti , B. Monsarrat , 1 and O. Burlet-Schiltz 1 Institut de Pharmacologie et de Biologie Structurale, CNRS, Université de Toulouse, Toulouse, France 2 Centre de Physiopathologie Toulouse-Purpan, INSERM U563, Toulouse, France Introduction arginine and lysine labelling allowed 90% of Proteasome is an essential component of the identified proteins to be quantified. Bioinformatic ubiquitin-proteasome pathway and plays a critical data analysis programs were then used to classify role in protein degradation regulation. Dysfunction proteins and to visualize pathways in both cell of this complex machinery can lead to various lines. Overall, the results obtained by combining pathologies including cancer (1). Proteasome different proteomic approaches for the inhibitors represent promising new antitumor drugs characterization of proteasome complexes as well and one of them is currently used for the treatment as the proteome content of these cell lines should of multiple myeloma. Our aim is to compare help understanding their differential anticancer proteasome complexes composition and activity drug sensitivity. and to analyze proteome variations in different leukemic cells to understand better the effect at Innovative aspects the molecular level of these drugs. • Analysis of proteasome complexes. • Quantitative proteome analysis of different Methods acute myeloid leukemia cells. Proteasome complexes were purified by • Structure/function relationships of proteasome immunoaffinity chromatography (2). Their subunits in acute myeloid leukemia cells. were separated by gel electrophoresis and identified by mass spectrometry. Proteasome References (maximum 3 references) activity was measured by following the hydrolysis (1) A. Mani et al., The ubiquitin-proteasome of fluorescent substrate peptides. Differential pathway and its role in cancer; J Clin. Oncol. 2005, quantitative proteome analyses were performed 23, 4776-4789. using stable isotope labeling with amino acids in (2) M.P. Bousquet-Dubouch et al, Purification 13 cell culture (SILAC) with labeled lysine ([ C6]Lys) and proteomic analysis of 20S proteasomes from 13 15 and arginine ([ C6, N4]Arg) amino acids. Mass human cells; Methods Mol. Biol. 2008 (in press). spectrometric analyses were performed by (3) D. Bouyssié et al, Mascot file parsing and nanoLC-MS/MS using either a Q-STAR or an LTQ- quantification (MFPaQ), a new software to parse, Orbitrap mass spectrometer. Database searches validate, and quantify proteomics data generated were performed using the Mascot algorithm and by ICAT and SILAC mass spectrometric analyses: quantitative data were analyzed using MFPaQ, a application to the proteomics study of membrane in-house developed software (3). proteins from primary human endothelial cells; Mol. Cell. Proteomics 2007, 6, 1621-1637. Results Two human acute myeloid leukemia (AML) cell lines showing differential sensitivity to proteasome inhibitors were studied: KG1a cells (AML M0) and U937 cells (AML M5). First, the proteasome status in each cell line was compared. We showed that the proteasome content and its chymotrypsin-like activity greatly differ in the two cell lines. After 20S proteasome purification, however, the subunit pattern observed by 2D gel electrophoresis revealed only minor differences in the subunit composition of the complex. Both standard 20S proteasome and immunoproteasome were present in both cell lines. Thus, regulatory complexes associated to the 20S catalytic core were also studied. Second, the proteomes of the two cell lines were compared using the SILAC quantitative approach. NanoLC-MS/MS analyses of nuclear, cytosolic and membrane protein fractions using an LTQ-orbitrap mass spectrometer led to the identification of more than 1000 proteins in each fraction. Protein quantification based on both MAPPING HUMAN PROTEIN INTERACTION NETWORKS: THE HUMAN PROTEOTHEQUE INITIATIVE (HUPI) 1 1 1 1 1 Benoit Coulombe , Philippe Cloutier , Annie Bouchard , Célia Jeronimo , Andrée-Anne Lacombe , Mathieu 2 2 3 1 Lavallée-Adam , Mathieu Blanchette , Jack Greenblatt , Diane Forget 1 2 Institut de recherches cliniques de Montréal, Université de Montréal, Montréal, Canada, McGill Centre for Bioinformatics, McGill University, Montréal, Canada, Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada Introduction performance mass spectrometry. Many identified Proteins are the central functional components interaction partners were targeted in reciprocal of human cells. They are involved in almost all tagging experiments in order to confirm many cellular processes, and protein aberrations have interactions and to enrich the dataset. High- been shown to have a causative role in many confidence interactions were selected diseases. Most importantly, proteins must interact computationally using an algorithm that we with other molecular components of the cell, developed and trained using machine learning to including other proteins, DNA sequences, RNA minimize the rate of both false-positives and molecules and various metabolites, to exert their false-negatives. functions. Mapping protein interaction networks in cells and tissues is expected to produce Results fingerprints of the physiological states of these The data produced with more than 100 affinity cells and tissues; similarly, mapping changes in tagged proteins was used to (i) build a high- protein interaction networks occurring during definition map of interactions that connect disease progression will generate signatures for components of the transcription and RNA specific pathological states. Because protein processing machineries in human cells; (ii) show interaction maps represent multi-variable, complex that transcription and RNA processing factors descriptions of physiological/pathological states, from the soluble cellular fraction are associated they also provide the descriptions needed to more with proteins that specifically regulate the realistically and reliably address issues such as formation (e.g. assembly, localization and/or the causes, the diagnoses and, eventually, the stability) of protein complexes; and, (iii) assign a cures of human diseases. Mapping protein putative function to a number of previously- interaction networks in health and disease is a uncharacterized proteins based on ''guilt by tremendous scientific and technological challenge association''. A number of previously- that will require huge efforts from many groups of uncharacterized proteins that we further scientists world-wide. characterized functionally and biochemically Recently, we have developed a technology define a novel class of regulatory factors that pipeline for the systematic characterization of target RNA polymerase II and other transcription protein interaction networks from human cells (1). factors prior and/or after the transcription reaction Our objective is to leverage this work into a large- on chromatin DNA. scale initiative aimed at building a repertoire of comprehensive maps of protein interaction Innovative aspects networks in health and disease. This repertoire of • High-precision mapping of human protein human protein interaction maps, that we propose interaction networks to name the Human Proteotheque, will be built via • Discovery of a novel class of cellular a concerted, international initiative that will involve regulatory factors that target the transcription a multi-site discovery platform aimed at defining machinery protein-protein, protein-DNA, protein-RNA and • Creation of the Human Proteotheque, an protein-metabolite interactions, and integrating the expanding repertoire of comprehensive maps data into comprehensive interaction maps through of human protein interaction networks in health bioinformatics. This effort, termed the Human and disease Proteotheque Initiative (HuPI) is at the heart of an emerging discipline, Integrative Systems Biology References (ISB), which is aimed at developing tools and (1) C. Jeronimo et al, Systematic analysis of concepts to generate complex descriptions of the protein interaction network for the biological systems considered globally. human transcription machinery reveals the identity of the 7SK capping enzyme; Mol. Methods Cell 27, 262-274, 2007 Here we used our newly developed technology pipeline to perform a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to high- Characterization of a novel subunit of the Drosophila melanogaster chromatin remodeling complex PBAP Gillian E. Chalkley 1, Yuri M. Moshkin 1, Karin Langenberg 1, Karel Bezstarosti 2, Andras Blastyak 4, Henrik Gyurkovics 4, Jeroen A. A. Demmers 2, and C. Peter Verrijzer 1 1. Department of Biochemistry, Center for Biomedical Genetics, and 2. Proteomics Center, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. 3. Hungarian Academy of Sciences, Biological Research Center, Institute of Genetics, H-6701 Szeged, Hungary Introduction consistent with the notion that SAYP is the key SWI/SNF ATP-dependent chromatin remodeling architectural subunit required for assembly of the complexes (remodelers) perform critical functions tri-partite PBAP module and its linkage to the in eukaryotic gene expression control. BAP and SWI/SNF core. PBAP are the fly representatives of the two In a genome-wide distribution analysis, SAYP co- evolutionarily conserved major subclasses of localized with both PB and BAP170 on larval SWI/SNF remodelers. Both complexes share 7 salivary gland polytene chromosomes on the core subunits, including the Brahma (BRM) majority of their chromosomal binding sites. These ATPase, but differ in a few signature subunits: sites also bind BRM and represent the interband Polybromo (PB) and BAP170 specify PBAP, regions of open, less condensed chromatin. These whereas OSA defines BAP. Here, we show using results suggest that PB, SAYP and BAP170 a proteomics approach that the transcriptional co- appear to form a stably associated PBAP-defining activator and PHD finger protein SAYP, is a novel module that is integrally recruited to chromatin. PBAP subunit. Furthermore, using biochemical Gene expression profiles from specific RNAi- and and genetic analyses, we give detailed insight into mock treated cells were compared, using an both the architecture and the function of the PBAP unbiased statistical analysis of the complete data holoenzyme. set. Correlation analysis of the SAYP transcriptome and the core, PBAP- and BAP- Methods selective subunits showed a very strong Polyclonal antibodies against BAP and PBAP relationship between SAYP and BAP170 and PB. subunits were raised and used for co- Principal component analysis revealed that the immunoprecipitation experiments from Drosophila SAYP profile is highly correlated with those of PB embryo nuclear extracts. Eluted proteins were and BAP170. resolved by SDS-PAGE and visualised by coomassie staining. Polypeptides were identified by mass spectrometry on an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fischer). Data analysis was performed using the Mascot search algorithm searching against FlyBase database. Further data analysis was performed using in- house developed software. For RNAi knockdown and genome wide expression analysis experiments, Drosophila S2 cells were cultured in Schneider's media and treated with double- stranded RNA for 4 days. RNA samples from three completely independent biological SAYP knockdown experiments were prepared and hybridized with Affymetrix microarrays. Results Mass spectrometric analysis revealed the presence of SAYP in the BRM- and PB-, but not in the OSA immunopurification. Stringent co- immunoprecipitation experiments showed that the Innovative aspects core subunits, BAP170 and PB co-purify in an anti- • Combination of mass spectrometry with SAYP immunoprecipitation, whereas OSA is biochemical and genetic analyses as a completely absent. Also, the association to the novel approach to the investigation of core by either OSA or the PBAP module was transcriptional pathways. found to be strictly mutually exclusive as no hybrid complexes were detected. Also, there seems to be References a hierarchy in PBAP architecture: SAYP is critical (1) Chalkley et al, Mol Cell Biol, Feb 25 [Epub for the integration of assembly. The results are ahead of print] DIFFERENTIAL BIOTINYLATION TO STUDY INFLUENZA A POLYMERASE SUBUNIT PB1 HETERODIMERISATION 1 2 2 Mathias Dreger , Tao Deng , and George Brownlee 1 Department of Physiology, Anatomy, and Genetics, University of Oxford, Parks Road, Oxford, OX1 3PT, United Kingdom 2 Sir William Dunn School of Pathology, University of Oxford, UK South Parks Road Oxford, OX1 3RE, United Kingdom Introduction: The influenza A virus polymerase complex, a heterotrimeric assembly of the subunits PB1, PB2, and PA, is critical for virus replication. PB1 requires PA and PB2 subunits for functional activity. To identify functionally important sites in PB1 sensitive to subunit interactions, we devised a strategy of SILAC labelling and biotinylation of solvent-accessible lysine residues to compare the biotinylation pattern of PB1 in its monomeric form or bound by the subunit PA. We identified a novel site crucial for PB1 function. Methods: TAP-tagged monomeric PB1 or PB1/PA dimers were purified from transiently transfected 12 13 HEK293T cells grown in C6-lysine- vs. C6-lysine-containing culture medium. Recovered proteins were biotinylated in the native state by an amine-specific reagent. The protein amount was estimated and the samples were combined 1:1, and separated by SDS-PAGE, followed by trypsin digestion. Biotinylated peptides were enriched by avidin affinity chromatography, and subjected to nanoflow LC. Fractions were spotted onto a target plate and analysed by Maldi-MS. SILAC pairs of biotinylated peptides were quantified. Lysine residues that displayed altered biotinylation between PB1 and PB1/PA were chosen for mutagenesis to assess their functional relevance for complex formation and polymerase activity. Results: The levels of PB1 in the PB1 monomer and in the PB1/PA dimer were similar. PA and PB1 were stoichiometrically present in the dimer sample. For mass spectrometric analysis, we first acquired data in automated nanoflow LC-Maldi MS/MS runs. In this workflow, a portion of the sample is still accessible on the target plate after the automated run. This allows to assign further candidate masses and to subject those peptides to a manually operated MS/MS analysis. The mass spectrometric analysis returned overall 19 biotinylated lysine residues of PB1, present in more than 30 different SILAC pairs. Six lysine residues displayed altered accessibility to biotinylation in the PB1 monomer vs. the PB1/PA dimer (changing 2- to 4-fold). Mutation analysis revealed that one residue that was less accessible in the dimer as compared to the monomer was found to be a novel site crucial for polymerase activity, but did not affect dimer formation. Another site displayed increased accessibility in the PB1/PA dimer, indicating a conformational change in PB1 upon PA binding. Our strategy is generally applicable to identify protein areas that are sensitive to protein-protein interactions. Innovative aspects: • a generally applicable strategy to semi-quantitatively assess changes in solvent exposure of protein surfaces was devised • the strategy may yield data on protein-protein interactions as well as on changes of protein conformation, and may identify functionally important sites of a protein • a novel site critical for PB1 polymerase activity was identified PB1 PA PB2 fig.1 the influenza A polymerase complex x10 4 Intens. [a.u.] 1303.754 2.5 2.0 1297.733 1.5 1.0 1325.730 1319.730 0.5 1313.739 00 1280 1290 1300 1310 1320 m/z fig. 2: SILAC pair of a biotinylated peptide Proteomics of Protein Complexes: An Approach to Analyze Native Proteomes 1 1 2,3 2 1 Oliver Drews , Yueju Wang , Emily Chen , John R Yates 3rd , and Peipei Ping 1 Department of Physiology, University of California Los Angeles, 675 CE Young Dr S, Los Angeles, CA 2 90095, USA; Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd, 3 SR11, La Jolla, CA 92037, USA; Department of Pharmacological Sciences, Stony Brook University, BST 8- 125, Stony Brook, NY 11974, USA Introduction assemblies and interactions can be retrieved, Proteins in cells translate signals via interactions, providing insights into physiology and elevating convert energy and metabolites in complexes, and proteomics to deliver a more comprehensive form structural networks in filaments, pores and analysis of proteomes. junctions. Proteins in proteomics though are predominately analyzed at large scale after Innovative aspects denaturation and hence viewed as singular • Native in-solution isoelectric focusing at high entities. In consideration of the fact that proteins resolution and the level of proteomes and interact in biological networks, we developed an subproteomes approach for large scale protein analysis while • Enables native two-dimensional maps of preserving proteins in their native state and most proteomes importantly maintaining the proteins in complexes. • Compatible with functional assays and online mass spectrometry Methods Challenging in analyzing a multitude of native References protein complexes is the separation at high (1) O. Drews et al., Mammalian proteasome resolution without perturbation of the fragile subpopulations with distinct molecular interactions between proteins. For our approach, compositions and proteolytic activities; Mol protocols for native isoelectric focusing of protein Cell Proteomics. 2007 Nov;6(11):2021-31. complexes by free flow electrophoresis were developed to achieve this goal. Furthermore, task oriented protocols were optimized and tested for compatibility with prevalent technologies used in proteomics. Thus, protein complexes were subsequently subjected to functional assays, further native or denaturing gel electrophoresis in a second dimension, and/or identified by online mass spectrometry. Results The approach was applied to native proteomes containing a widely different number of protein complexes to demonstrate its effectiveness at various scales. Separation of crude extracts from S. cerevisiae consisting of a large variety of complexes was achieved at a resolution of 0.1 pH units between pH 3-10. Interestingly, the complexes focused between pH 4.5-7.5, sharing only in part the band-with of pIs of denatured yeast proteins. Subsequent native PAGE provided a 2-D map of yeast complexes, showing protein bands from 50-1000 kDa. Identification by MS confirmed the separation of known and hitherto unknown homomeric as well as heteromeric complexes. For example, the homohexamers His1 and Gdh1 separated at 200 and 300 kDa, and focused at pH 5.4 and 5.2 in close proximity to their theoretical pIs 5.87 and 5.56. At the other end of the dynamic range, proteasome complexes, purified to apparent homogeneity, were separated in subpopulations at a resolution of 0.04 pH units (1). With the approach, novel information about protein Topology and dynamics of protein interaction networks that control cell growth Oliver Rinner, Timo Glatter, Alexander Wepf, Katja Köhler, Irena Jevtov, Hugo Stocker, Ernst Hafen, Ruedi Aebersold and Matthias Gstaiger Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland Introduction interaction networks; Nat. Biotechnol. The control of cellular growth under changing 2007 environmental conditions (oxygen saturation state, presence and concentration of growth hormones and nutrients etc) is an essential physiological process with important clinical implications. To date, the response of cells to changes in their environment has been primarily studied using the methods of cell biology, biochemistry or genetics in which complex cellular responses are dissected into linear sequences of signaling events. Here we present a systems oriented approach to study a protein interaction network for the control of cell growth control. QuickTime™ and a Methods TIFF (LZW) decompressor are needed to see this picture. Building on the results of genetic screens in Drosophila melanogaster for genes affecting cellular growth, and by applying advanced quantitative mass spectrometry techniques we characterize protein interaction networks in response to the growth hormone insulin in drosophila KC167 cells. Protein complexes are crosslinked with DSP prior to affinity purification using a single step immuno-affinity purification approach. Interacting proteins are identified by Figure 1. Evolutionary conservation of complexes containing the target of rapamycin (TOR) kinase direct LC-MS/MS and relative changes in protein between D.melanogaster and humans. interactions are quantified using the recently developed "MasterMap" concept (1). Results We have generated a collection KC167 cell lines for the inducible expression of epitope tagged bait proteins. Following affinity purification and mass spectrometry (AP-MS) we identified a number of specific protein interactions that are highly conserved between humans and D. melanogaster, including complexes containing the PIK-related kinase dTOR (Figure1). Relative quantification from aligned MS1 spectra uncovered a set of insulin dependent interactions within the drosophila insulin receptor/target of rapamycin (InR/TOR) signaling pathway. These hormone dependent interactions may provide important cues for understanding the molecular mechanisms underlying insulin dependent growth control. Innovative aspects • Combined genetics and mass spectrometry approach • Evolutionary conservation of protein complexes • Hormone dependent protein-protein interactions References (1) O.Rinner et al., An integrated mass spectrometric and computational framework for the analysis of protein Pull-downs of endogenously expressed, tagged proteins for interactions and modifications 1 1 1 1 1 1 Nina C. Hubner , Johannes Graumann , Michiel Vermeulen , Leonie Waanders , Jürgen Cox , Yong Zhang , 2 2 2 1 Alexander Bird , Ina Poser , Anthony Hyman , Matthias Mann 1 Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany 2 Department for Microtubules and Cell Division, Max-Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Str. 108, 01307 Dresden, Germany Introduction way from a minimal amount of protein. Briefly, we Multi-protein complexes and modifications are obtain very pure bait protein by using more fundamental in most cellular processes. It is of stringent washing conditions and perform in-depth great interest to the scientific community to provide sequencing and assignment of all its peptides. a network of interactions and modifications. While there exists already a large-scale interaction Innovative aspects dataset for yeast, it has so far not been possible to • Interaction studies with endogenous map mammalian interactions and modifications to expressed proteins in mammalian systems a similar depth. • High-confidence determination of interaction Our goal is to establish a standardized pipeline for partners and modifications using SILAC mapping specific protein interactions and • Unbiased identification of modifications modifications by SILAC-based quantitative mass spectrometry (Mann, 2006). References Mann, M., "Functional and quantitative proteomics Methods using SILAC", Nature reviews 7, 952-958 (2006) Proteins are tagged by homologous recombination Muyrers, J.P et al., "Techniques: Recombinogenic under their endogenous promoter with GFP in engineering - new options for cloning and HeLa or embryonic stem cells (Muyrers, 2001). In manipulating DNA", Trends in biochemical contrast to published large-scale interaction sciences 26, 325-331 (2001). studies in mammalian systems we do not Poser, I. et al., "BAC TransgeneOmics: A high- overexpress the bait protein and thereby keep the throughput method for exploration of protein system in a close to natural state. We combine a function in mammals", submitted highly specific antibody to the GFP-tag with very small magnetic beads and a novel HPLC technique, which allows analysis of very low amounts of material and repeated and targeted measurement. Using this system with the LTQ- Orbitrap even low abundance proteins from only one 15cm cell culture dish can be analyzed. SILAC is used to distinguish true interaction partners from background binders. Results We demonstrate pulldowns from SILAC-encoded tagged and control HeLa cell lines. Tagged cell lines are labeled with heavy arginine and lysine, An empty control cell line or another tagged cell line is labeled with light arginine and lysine. Peptides derived from the two samples can be distinguished by MS owing to their mass difference and the signal ratio between SILAC pairs directly indicates the protein abundance ratio. Unspecific background binders show a ratio of 1:1 as they are equally present in both samples. The bait protein Figure 1. The histogram and boxplot show protein ratios and specific interactors will have a ratio different of obtained from a co-immunoprecipitation of GFP-tagged 1 as they are only present in either light or heavy gamma tubulin (heavy SILAC amino acids) and HeLa wt from. For tubulin gamma we identified all known as a control (light SILAC amino acids). Background binders are normally distributed (B). Outliers are either interactors with high ratios and found one protein contaminants like trypsin or human keratins introduced with high confidence that is not yet known to be a during sample preparation (A) or true tubulin gamma gamma tubulin binder. interactors like tubulin-gamma complex components 2-6 We also introduce a method to identify even and T-complex proteins 1-8 (C and encircled) that show underrepresented modifications in an unbiased very high heavy/light ratios. "Tagless" Strategy of Protein Complex Identification: Towards High Throughput 1 2 1 2 1 1 1 Lee Yang , Haichuan Liu , Ming Dong , Simon Allen , Peter Walian , Bing Jap , Terry C. Hazen , Steven C. 2 1,2 1 1 2 1 Hall , Susan J. Fisher , John-Marc Chandonia , Mark D. Biggin , H. Ewa Witkowska , Jian Jin 1 2 Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Cell and Tissue Biology, University of California, San Francisco, CA 94143, USA. Introduction developed to enable information-based non- A novel "tagless" strategy for protein complex redundant MS/MS acquisition. The integration for identification from native preparations has been fully automated operation is under way. proposed (1). Putative protein complexes are identified based on the MS-monitored co-migration Innovative aspects of collections of polypeptides through multiple • Identification of protein complexes under orthogonal protein separation steps. As part of the native conditions without genetic Genomics: GTL Protein Complex Analysis Project manipulation or affinity tags. we are developing a high throughput pipeline to • Fully integrated and automated system for automate the processes of sample preparation, instrument control, intelligent MS/MS MS data acquisition and interpretation to enable acquisition, data processing and analysis of thousands of fractions required for a management. comprehensive organism-wide mapping of bacterial interactomes. References 1. Dong et al., "A "Tagless" Strategy for Methods Identification of Stable Protein Complexes Cell lysates undergo a 4-step protein separation Genome-wide by Multi-dimensional process (ammonium sulfate precipitation, SCX, Orthogonal Chromatographic Separation TM HIC and SEC chromatography). Fractions and iTRAQ Reagent Tracking". J. collected at the last step are digested with trypsin, Proteome Res. 2008 (in press) labeled with iTRAQ and analyzed by MS/MS using a LC MALDI workflow. Polypeptides are identified and their elution profiles are derived on the basis of iTRAQ-based relative quantitation. Cluster analysis of the data groups polypeptides with similar elution profiles into putative protein complexes. Methods are being developed for high-throughput sample processing (digestion and iTRAQ labeling), automated MS with a feed-back loop for generating intelligent information-based iterative MS/MS acquisition routines and automated submission of polypeptide ID and quantification data to clustering analysis. Results The current scale of biomass production is 400 L of D. vulgaris (10 g soluble protein). Comprehensive characterization of the D. vulgaris interactome necessitates analysis of thousands of protein fractions. Development of modules and tools required for automating the sample and data processing is being pursued. To date, 0.2% of protein separation space has been analyzed resulting in detection of at least 6 heteromeric and 45 homomeric complexes. Identified bottle-necks are being addressed: sample preparation, time efficiency of MS data acquisition and data management. Protein digestion and labelling are performed on PVDF membrane in a 96-well format. MS data acquisition, database search, generation of polypeptide elution profiles and clustering analysis can all be scheduled and processed in batches. Algorithms for analyzing and comparing precursors, retention time predictions and exclusion list creation are being Characterization of protein-complexes complementing functional-genomic screens in human cell-lines Magno Junqueira; Yusuke Toyoda; Mikolaj Slabicki; Zoltan Maliga; Mirko Theis; Dragomir Krastev; Frank Buchholz; Antony Hyman and Andrej Shevchenko Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Introduction on statistical considerations. In 93% of Here we report on a rapid and sensitive pipeline experiments we recovered the bait as one of top for the systematic characterization of human hits and in 74% we found potential interaction protein complexes, which employs BAC partners from which 20% have been confirmed by expression of tagged proteins, digestion of affinity other techniques. purified proteins directly in large volumes of eluate from affinity column, off-line concentration-cleanup Innovative aspects of peptides and their accurate identification via • Identification of 70 new native mammalian single LC-MS/MS run on a LTQ Orbitrap protein complexes instrument. • Affinity isolation of endogenous mammalian complexes is an efficient orthogonal assay that Methods complements esiRNA loss-of-function and BACs with the localisation and affinity purification localization screens. (LAP) cassette containing GFP and S-peptide sequences were inserted as carboxy-terminal fusion and transfected in HeLa cells to obtain stable expression. Human protein complexes were purified by affinity chromatography using an anti- GFP antibodies and digested with trypsin directly in the elution buffer containing 100 mM glycine in 100 mM Tris pH 8.0. Tryptic peptides eluted in up to 0.5 mL volume were cleaned up and concentrated off-line on UltraMicroSpin C18 cartridge and then sequenced by LC-MS/MS on a LTQ Orbitrap instrument using 145 min elution gradient (5 to 60 % of acetonitrile in 0.1% formic acid). In house developed scriptswere applied to recognize and validate the interacting partners. Results Figure 1- Comparative workflow for direct LC-MSMS We demonstrate that, because of the high spectra (shotgun) and for GeLC-MSMS. The new method allows acquisition rate and dynamic range of LTQ 20 times faster analysis when compared with GeLC- Orbitrap, no 2D-LC separation was required to MSMS. dissect the composition of protein complexes. Our method provided 20x faster analysis with equal or better performance compared to Gel-LC-MS/MS approach – a recognized "golden" standard in the field. We applied the method to a large series of baits selected via genome-wide esiRNA loss-of-function screens. Knock-down phenotypes usually do not immediately reveal the unequivocal functional assignment of targeted genes. However, clues on protein complex function could be inferred by following "guilty by association" concept, using known function or characteristic sequence domains of identified interaction parteners Within 15 months more than 300 pull-downs were Figure 2- APC-complex was isolated by TAP (CDC16 screened for more than 70 individual genes under "Bait") and the sample was split into two equal parts. different experimental conditions. Each experiment One was analyzed by GeLC-MS/MS, while another one typically produced over 250 different proteins, was digested in-solution and directly analyzed by direct while ~80% were sorted out as background based LC-MSMS. The chart shows the sequence coverage for all subunits achieved by both methods. Chromatin Proteome Demonstrates System-Wide Response To Replication Inhibition # § Guennadi A.Khoudoli*, Peter J. Gillespie*, Graeme Stewart , Jens S. Andersen , Jason R. Swedlow*and J. Julian Blow* * - University of Dundee, College of Life Sciences, Dow Street, Dundee, DD1 5EH, Scotland, UK, e-mail: email@example.com Introduction to progression through replication (Figure 2). The Stable and accurate propagation of chromosomal data are consistent with the idea that Mcm2-7 DNA during cell division cycle is a fundamental licensing complex plays a central role in coordi- function of all living organisms. The pathways nating nuclear structure with DNA replication [3]. regulating replication, expression and maintenance of genetic information are well studied. However, Innovative aspects the way that these different processes work • Functional modules on replicating chromatin together as a coordinated biological system is and their temporal order poorly understood. Here we report a • System-wide effect of replication inhibition on comprehensive analysis of the changing the dynamics of the chromatin proteome association of proteins with chromatin (the • Central role of Mcm2-7 licensing complex in chromatin proteome) during progression through coordinating nuclear structure with DNA interphase of the cell cycle in the absence or replication presence of replication inhibitors. References: Methods 1. Futschik, M.E., and Carlisle, B. (2005). J Nuclei were assembled in vitro in Xenopus cell Bioinform Comput Biol 3, 965-988. free extracts and then purified at defined points 2. Dennis, G., at al. Genome Biol 4, P3. during the progression through interphase during 3. Khoudoli, G.A. at all (Prepared for publication) replication, as well as in the presence of replication inhibitors geminin or roscovitine. Associated proteins were eluted from chromatin, digested with trypsin and subjected to LC MS/MS analysis. The proteins abundance was estimated from the extracted ion chromatograms (XIC) of their corresponding peptides. Using fuzzy c-mean clustering (FCM) [1] proteins were grouped into 12 clusters based on the dynamics of their association with chromatin. The DAVID software [2] was used to collect ontological terms associated with all the proteins and to determine if any of them were enriched in specific FCM clusters. Figure 1. FCM clustering of temporal profiles of polypeptides associated with replicating chromatin Results The final set of non-redundant proteins subjected to the analysis consisted of 746 entries. Of the 606 proteins identified on untreated chromatin, 458 demonstrated more than 30% variation in abundance during interphase. 12 clusters generated by FCM algorithm were divided into three general types that have their peak abundance on chromatin in early, intermediate or late interphase (Figure 1). Annotations analysis demonstrated that FCM clusters identify a series of modules consisting of functionally-related protein factors that associate with interphase chromatin in a defined order. Figure 2. Hierarchical clustering of the combined data We examined the effect of blocking DNA set. replication on the chromatin proteome by inhibiting either replication licensing or S phase CDK # - . Biomedical Research Centre, University of activity. This revealed an unexpectedly broad Dundee, Ninewells Hospital and Medical School, system-wide effect on the dynamics of the Dundee. DD1 9SY, UK chromatin proteome. The behaviour of functional § - Center for Experimental BioInformatics, groups reflected a range of different nuclear University of Southern Denmark, Campusvej 55, functions that are synchronised with and respond DK-5230 Odense M, Denmark Quantitative Analysis of C/EBPα Transcription Complexes in Leukemia Rositsa I. Koleva, Scott B. Ficarro, Manor Askenazi, Jarrod A. Marto Department of Cancer Biology, Dana-Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 44 Binney St., Boston, MA 02115, USA Introduction Acute myeloid leukemia (AML) remains a highly Innovative aspects lethal malignancy with limited therapeutic options. • Novel multidimensional fractionation with true Mounting evidence indicates that impaired function nanoflow chromatography but not complete absence of the transcription • Quantitative analysis of protein complexes factor C/EBPα leads to consecutive accumulation responding to molecular switch of myeloid progenitors and blast crisis in AML. • Target protein validation in myeloid Recent studies demonstrated that oncogenic Flt3 differentiation model signaling leads to phosphorylation on serine 21 (pS21) of C/EBPα with concomitant impairment of References myeloid differentiation [1, 2]. We hypothesized that (1) Radomska, H.S., et al., Block of C/EBPα function by assembly of transcriptionally active protein phosphorylation in acute myeloid leukemia with complex is governed by pS21-regulated protein FLT3 activating mutations. J. Exp. Med., 2006. associations. 203(2): p. 371-381. (2) Ross, S.E., et al., Phosphorylation of C/EBPα Inhibits Granulopoiesis. Mol. Cell. Biol., 2004. 24(2): Methods p. 675-686. To test this hypothesis we established affinity tagged C/EBPα expression system under control of a tet-inducible promoter in myeloid cells with constitutive Flt3 activity. Treatment of these cells with Flt3 inhibitors modulated pS21 in a dose- dependent manner. Next, advanced quantitative proteomics methodology, including true nanoflow LC coupled with multidimensional RP/RP fractionation and on-column iTRAQ stable isotope labeling, was used to monitor remodeling of C/EBPα protein complexes as a function of pS21. siRNA methods were used to abrogate expression of interactors modulated by pS21 to determine their functional role in transcription of myeloid- specific genes. Figure 1. C/EBPα putative partners' network. The edges Results reflect HPRD data base annotated protein-protein Our data represent by far the largest catalog interactions assessed by various experimental C/EBPα interactors, including more than 200 approaches. The network is enhanced by addition of proteins involved in chromatin organization, new edges (in light green) based on manual curation of transcriptional modulation, and cell cycle recent literature. Target proteins selected for further regulation. Furthermore, our quantitative studies are highlighted. proteomics data demonstrate that 1) C/EBPα 9.0 C/EBPα activation interacts with proteins genetically linked to 8.0 7.0 leukemia; and 2) many of these interact with 6.0 C/EBPα in a phosphorylation-dependent manner. 5.0 Knock-down of newly-identified, leukemia- 4.0 associated interactors (targets #1-4) reduced the 3.0 2.0 ability of C/EBPα to drive expression of 1.0 granulocytic target genes. Our data demonstrate 0.0 that phosphorylation on serine 21 modulates Control A B Sub siRNA 1 Sub siRNA ( A Meis1#1 B Meis1#3 Control 2) association of C/EBPα with protein partners that siRNA Target #1 Target #2 are functionally relevant for myeloid differentiation. siRNA siRNA Our ability to quantitatively monitor multiple leukemia-related gene products in the context of Figure 2. Depletion of target genes #1 and #2, C/EBPα protein complexes provides valuable corresponding to novel C/EBPα partners, decreases insight into the mechanisms by which oncogenic C/EBPα capacity to induce the transcription of kinase activity disrupts transcription and leads to granulocytic genes in myeloid cells. leukemogenesis. Shot-gun membrane proteomics by acid hydrolysis combined with trypsin and chymotrypsin 1 1 2 1 1 Joseph Kown , Jeehyun Oh , Sunghoon Lee , Seung Il Kim , and Jong-Soon Choi 1 Proteome Research Team, Korea Basic Science Institute, Daejeon 305-333, Korea 2 Korean Bioinformatics Center, KRIBB, Daejeon 305-806, Korea Introduction helix proteins, resulting in a high coverage level of Proteomics of membrane proteins is essential for membrane proteins. The predominantly expressed the understanding of cellular function. So far, mass proteins by the relative abundance indexing spectrometric analysis of membrane proteome has revealed the components of phycobilisome been done primarily in the identification of soluble complex (CpcA, CpcB, ApcB, ApcA, ApcE, CpcD), proteins. In order to attain comprehensive PS-II complex (PsbX, PsbY, PsbK, PsbE, PsbD1), membrane proteome in a cell, the well-developed PS-I complex (PsaF, PsaM, PsaB) and complex V sample preparation protocol is required for solving (AtpG). These are notably involved in the membrane hydrophobicity. Both gel-based photosynthesis by capturing light energy. analysis and shot-gun mass spectrometry method can be used for membrane proteome analysis. Innovative aspects Here we challenged to analyze the whole • Acid hydrolysis of multiple trans-membrane membrane proteome of cyanobacteria by acid proteins at aspartic acid for better identification hydrolysis combined with trypsin and chymotrypsin. • Acid hydrolysis followed by either trypsin or Cyanobacteria are a model photosynthetic chymotrypsin treatment for membrane proteins microorganism that is capable of energy • Massive identification of membrane proteins generation and metabolite transport via multiple- by shot-gun membrane protein profiling layered membranes and associated proteins (1). MudPIT Shot-gun membrane proteomics can give a more concrete data in mapping the repertoire of References cyanobacterial membrane-linked proteome. (1) F. Huang et al, Proteomics of Synechocystis sp. strain PCC 6803; Mol. Methods Cellular Proteomics. 2002 1, 956-966. Using a whole proteome scale prediction method, (2) C. C. Wu and J. R. Yates III, The 768 proteins were found to be present in the application of mass spectrometry to genome of unicellular cyanobacterium membrane proteomics; Nature Biotech. Synechocystis sp. PCC 6803. We employed an 2003 21, 262-267. experimental method to verify the membrane proteome en masse. A shot-gun membrane A B proteomic technique was used in combination of acid hydrolysis at aspartic acid with trypsin or chymotrypsin by multi-dimensional protein identification technology (MudPIT) (2). Results A total of 472 proteins were successfully identified from 1840 unique peptides and 153 proteins among them were membrane-linked proteins such as extracellular [3], outer membrane [11], periplasmic [7], plasma membrane [98], and membrane complex [34] were identified by Synechocystis sp. PCC 6803 proteome database. Figure 1. Size distribution and predicted region of In particular, 134 integral membrane proteins identified peptides resulting from chemical-trypsin (IMP) with transmembrane helices were detected method (CT) and chemical-chymotrypsin method (CC) by Phobius and Soshui prediction methods. Peptide masses are plotted at 500 Da unit. The Among these, the majority of detected IMP was distribution of peptides produced by the theoretical done by the chemical digestion with chymtotrypsin prediction is compared to that produced in experiment [100/134] rather than trypsin [52/134]. As expected (B). Each peptide is annotated the portion of signal in the mass range prediction of peptides cleaved peptide (pale blue), extracellular region (red), chemically and enzymatically from whole transmembrane region (yellow) and cytoplasmic region membrane proteome, the high frequency of mass (blue) as a color. range 1500~3000 generated by chymotrypsin after chemical digestion was available for MS/MS analysis to identify the multi-spanning membrane Proteomics study on human Ccr4-NOT: a multi-functional complex involved in mRNA metabolism 1 2 1 2 1 Nga-Chi Lau , Annemieke Kolkman , W.W.M. Pim Pijnappel , Albert J.R. Heck , and H. T. Marc Timmers 1 Department of Physiological Chemistry, University Medical Center – Utrecht, Universiteitsweg 100, 3584 CG 2 Utrecht, The Netherlands; Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands Introduction Results The Ccr4-NOT complex consists of multiple CNOT The obtained datasets revealed that not all CNOT proteins and is evolutionarily conserved from yeast proteins are stable components of the human to mammals. Its CNOT subunits have distinct Ccr4-NOT complex. Also, new subunits of the functions in synthesis and degradation of mRNA complex have been identified as well as other molecules (figure 1). A developmental role is also interesting novel interactors. In addition, several indicated for CNOT7 in mouse spermatogenesis distinct Ccr4-NOT complexes have been identified. and for CNOT1 in early development of C.elegans. Future experiments will be focused on the Here we describe a comprehensive proteomics functional differences between these Ccr4-NOT study in human cells to identify the composition complexes in human cells. and cellular interactors of the whole Ccr4-NOT complex in order to understand its functioning in cells. Innovative aspect • Combining epitope-tagging in human cells, protein ubiquitylation mRNA degradation double affinity purification and mass (E3 ubiquitin ligase) (deadenylation) spectrometry not only identified novel components of macro-molecular complexes, CNOT4 but also revealed distinct complexes differing CNOT3 in subunit composition CNOT7 CNOT6 CNOT2 CNOT1 CNOT8 CNOT9 mRNA synthesis (transcription) Figure 1. Human Ccr4-NOT complex components (CNOT proteins) are involved in synthesis (yellow) and degradation (green) of mRNA molecules. CNOT4 may play a role in protein ubiquitination, and CNOT3 has an unknown function. Methods Clonal stable human cell lines expressing near endogenous levels of epitope-tagged CNOT proteins were created. Associated proteins of a tagged subunit were purified from a cell line via a two-step affinity purification protocol (flag-HA), followed by in-solution trypsin digestion and LC- LTQ-FT MS/MS mass spectrometry for identification. Data integration of all CNOT purifications resulted in a protein network centered on the Ccr4-NOT complex components. Identification of protein phosphorylation sites associated with protein complex assembly using an IMAC-based proteomics procedure 1 1, 2 Chang-Hung Lee and Yeou-Guang Tsay 1 2 Institute of Biochemistry and Molecular Biology and Proteomics Research Center, National Yang-Ming University, Taipei, Taiwan Introduction It is a common belief that protein modifications, like phosphorylation, may modulate protein complex metabolism and thus effectively regulate protein functions. However, there are only a limited number of known examples thus far and most of them are uncovered via the conventional molecular biology approach. Methods Here we demonstrate the design and application of a new proteomics platform that aims to identify protein phosphorylation sites that are associated with protein complex assembly. We first undertake gel filtration liquid chromatography to resolve the protein complexes from Hela cell at their native 3+ states. Immobilized ferric ion (Fe ) affinity chromatography is then employed to enrich the phosphorylated peptides in each gel filtration fraction, whose identities and the phosphoamino acid residues within are documented using liquid chromatography-tandem mass spectrometry along with computer programs like TurboSequest and our in-house software. In order to optimize the isolation method for phosphopeptides, we have tested a multitude of the experimental conditions and systematically examine their impacts on the specificity and comprehensiveness of the procedure. Results Among various factors, we found that the pH and organic solvent in wash and elution solutions appeared to be particularly important. With this platform, we have identified a group of phosphorylation sites from HeLa cells that seem to be only present in the high-MW protein complexes, but not in the low-MW ones. Innovative aspects • Development of a new proteomics platform • Optimization of IMAC procedure for phosphopeptides purification • Identification of protein phosphorylation sites associated with protein complex assembly Molecular recognition in oligomeric enzymes: integration of bioinformatic and biosensoric approaches Yu. Mezentsev, A. Lisitsa, P. Ershov, A. Molnar, A. Ivanov, A. Archakov V.N. Orechovich Institute of Biomedical Chemistry RAMS Pogodinskaya str. 10, Moscow, 119121, Russia Introduction This work was supported in part by Russian Molecular recognition in protein complexes plays a Foundation for Basic Research (grant 07-04- central role in biochemical processes. It 00575). understanding is an important task in different Innovative aspects fields of biomedical science and drug discovery. Combination of bioinformatic and biosensoric The interface areas of protein-protein interactions methods in protein oligomerization research. have unique structures and represent prospective References targets for a new generation of drugs. The most (1) Mezentsev Yu.V., Molnar A.A., Gnedenko O.V., interesting group of similar targets are oligomeric Krasotkina Yu.V., Sokolov N.N. and Ivanov enzymes. This report shows integrative approach A.S. Oligomerization of L-Asparaginase from using bioinformatic and biosensoric methods to Erwinia carotovora. Biochemistry (Moscow) research of molecular recognition in oligomeric Supplement Series B: Biomedical Chemistry. enzymes. 2007, 1(1), 58–67. Methods (2) Ivanov A.S., Gnedenko O.V., Molnar A.A., We have chosen two oligomeric enzymes as test Mezentsev Y.V., Lisitsa A.V., Archakov A.I. molecular objects most convenient for such Protein-protein interactions as new targets for research — HIV-1 protease (HIVp) (homo-dimer) drug design: virtual and experimental and bacterial L-asparaginases (homo-tetramer) [1]. approaches. J Bioinform Comput Biol. 2007, We have used some computer methods 5(2b), 579-592. (molecular modeling, computational alanine scanning, molecular dynamics simulation and energy optimization), as well as direct molecular interaction measurement by SPR-biosensor. All calculations were done using Sybyl 6.9.1 (Tripos Inc.) software running on SGI Origin200 server and Amber 7. Biosensor measurements were carried out on Biacore-3000. Results Computer analysis of subunits contact areas in HIVp dimer was done using virtual «alanine scanning». Several amino acid residues which bring the significant contribution into the interaction energy ("hot spots") have been found. Also such analysis of subunits contact areas in homotetramers of bacterial L-asparaginases was done. The basic attention was given the interface between dimers AC and BD and between monomers in these dimers. It was shown, that in each subunit there are 13 residues which play a key role in interaction between dimers AC and BD. We modeled chimeric tetramers by substitution of one subunit on subunit from another L- asparaginase. The value of calculated interaction energy decreased. We tested this results in experiments on optical biosensor. It was shown, that the interaction between subunits from different L-asparaginases is impossible, even between subunits with high similarity (sequence identity >90%). Thermodynamics of subunits interaction in both enzymes, as well as interaction inhibitors of HIVp dimerization [2] was also studied using optical biosensor. CAPTURE OF THE ACTIVATED FC RECEPTOR COMPLEX FROM THE SURFACE OF LIVE CELLS BY||AFFINITY RECEPTOR CHROMATOGRAPHY Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured by the from the surface of live cells using microbeads coated with the receptor's cognate ligand (IgG) and analyzed by LC-MS/MS alongside several controls. Live-cell Affinity Receptor Chromatography (LARC) resulted in a partially non-redundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by previous genetic and biochemical methods and permitted the discovery of novel complex members. Confocal microscopy was used to confirm recruitment of specific members of the Fc, SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex including RHOG, P115 RHOGEF, and CRKL. The recruitment of AKT PH domain GFP was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the cell surface of live cells using ligand coated microbeads for identification of members of a receptor complex or pathway by liquid chromatography and tandem mass spectrometry. Post-Translational Modifications of the Endogenously Expressed Transcription Factors TFIID and SAGA 1 1 2 2 1 N. Mischerikow , A.F.M. Altelaar , H.T.M. Timmers , W.W.M. Pijnappel & A.J.R. Heck 1 Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University, Sorbonnelaan 16, 3584 CA 2 Utrecht, The Netherlands; Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Introduction References The general transcription factor TFIID and the (1) Sanders et al., Proteomics of the Eukaryotic histone acetyl transferase SAGA are multi-subunit Transcription Machinery: Identification of protein complexes in Saccharomyces cerevisiae Proteins Associated with Components of that have a number of subunits in common. Up to Yeast TFIID by Multidimensional Mass now, most proteomic studies have led to a static Spectrometry. Mol Cell Biol, 2002 picture of these complexes (1). However, their (2) Puig et al., The Tandem Affinity Method: A subunit composition may vary with time as a General Procedure of Protein Complex mechanism for transcriptional regulation. In this Purification. Methods, 2001 project we compare the post-translational state of affinity purified TFIID and SAGA. The aim is to comprehensively identify post-translational modifi- cations (PTMs) that might regulate the dynamics of components shared between the complexes. Methods TFIID and SAGA were tandem affinity purified from yeast whole cell extract (2) and precipitated. Both complex preparations were analyzed by SDS PAGE, digested in gel with Trypsin and analyzed by LC-MS. Additionally, both preparations were also digested in solution with Lys-C/Chymotrypsin, Lys-C/Glu-C and Lys-C/Trypsin, followed by SCX chromatography and LC-MS. Peptide analysis was realized using nano-LC coupled online to a LTQ- Orbitrap. Peptides and proteins were identified using the Mascot database search engine. Results The generic tandem affinity purification in combi- nation with tryptic in gel digestion allowed the iden- tification of all subunits commonly ranked among TFIID and SAGA with good average sequence co- verage. However, in some of the 5 TBP-associated factors (TAFs) present in both TFIID and SAGA, sequence coverage was relatively low. Moreover, in these and other subunits segments of sequence of functional importance were poorly covered. We therefore followed a multiple protease approach to enhance sequence coverage as a prerequisite to comprehensively map PTMs. Using this approach, we were able to identify a number of different PTMs on both complexes which to our knowledge have not been reported. Innovative aspects • comprehensive description of different types of PTMs of yeast TFIID and SAGA • comparison of PTMs on subunits shared between yeast TFIID and SAGA Spatiotemporal Changes of PKA-AKAP Complexes in Response to Prostaglandin E2. 1 2 1 2 1 Nikolaus G. Oberprieler , Thin Thin Aye , Knut Martin Torgersen , Albert J.R. Heck , Kjetil Taskèn 1) Biotechnology Centre of Oslo, University of Oslo, N-0317 Oslo, Norway 2) Department of Biomolecular Mass Spectrometry, Utrecht University, 3584 CA Utrecht, the Netherlands Introduction PKA-AKAP signalling complexes and provide new Intracellular signalling events play a crucial role in insights into the spatiotemporal regulation of PKA- T-lymphocytes under physiological and AKAP signalling complexes in T-lymphocytes. pathological conditions. Prostaglandin E2 (PGE2) signalling is important in the negative regulation of Innovative aspects T-cell receptor signalling and increased PGE2 • Chemical proteomics approaches lead to the signalling has been implicated in diseases such as successful enrichment of PKA-AKAP HIV infection and cancer. PGE2 signalling is complexes. primarily mediated through cAMP and the • A combination of molecular biology and subsequent activation of PKA. In order to allow proteomics approaches revealed novel PKA- targeted action of PKA the kinase is anchored to AKAP signalosome members. macromolecular signalling complexes by protein • Novel aspects of the spatiotemporal regulation kinase A anchoring proteins (AKAPs). of PKA signalling were revealed. Methods 8-AHA-cAMP-agarose beads were used to enrich cAMP binding proteins and their secondary binding partners. This resulted in a significant enrichment of PKA regulatory subunits and their anchoring proteins (AKAPs). For quantitative proteomic analysis, peptides from agarose bead fractions were dimethyl labelled and subjected to ESI- FTICR MS. Additionally, identified AKAPs were expressed in SILAC labeled Jurkat T-cells as FLAG-tagged proteins. Following expression, AKAP complexes were purified using anti-FLAG antibody covalently linked to magnetic Dynabeads. Isolated AKAP complexes were analysed by LC- orbitrap MS. Furthermore, GFP-tagged AKAPs were expressed in Jurkat T-cells to determine changes in AKAP localization following PGE2 treatment using fluorescent microscopy. Results The chemical proteomics approach using 8-AHA- cAMP-agarose beads identified 6 AKAPs which are expressed in primary human T-lymphocytes. Furthermore, quantitative analysis of samples stimulated with PGE2 for 10 and 60min revealed temporal regulation of PKA-AKAP complexes. Several proteins were identified which either get recruited to PKA-AKAP complexes or dissociate Figure 1: Primary T-lymphocytes were stimulated with following PGE2 treatment. In order to determine PGE2 (10μM) for 10 and 60min and compared to the composition of AKAP-specific signalling unstimulated controls (CT). Cells were lysed and cAMP- binding fractions enriched using 8-AHA-cAMP-agarose complexes, the identified AKAPs were individually beads before subjecting each sample to SDS-PAGE and expressed as FLAG-tagged proteins in SILAC Coomassie blue gel staining. Arrows highlight proteins labelled Jurkat T-cells. This allowed the which are recruited or dissociate with PGE2 treatment. identification of several AKAP-specific signalling- complex members and revealed their temporal regulation in response to PGE2 treatment. Again, a number of proteins were specifically recruited or dissociated from AKAP signalling complexes. The expression of GFP-tagged AKAPs in Jurkat T-cells allowed the analysis of spatial regulation of AKAP complexes in response to PGE2 treatment. Taken together, the results identify novel members of LOOKING FOR E2F2 TRANSCRIPTION FACTOR INTERACTING PROTEINS 1 2 3 3 1 Nerea Osinalde , Kerman Aloria , Ana Zubiaga , Asier Fullaondo and Jesus M. Arizmendi 1 Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Spain. 2 Proteomics Unit SGIker, University of the Basque Country, Leioa, Spain 3 Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Leioa, Spain Introduction Now these potential E2F2 interacting partners are E2F2 is member of the E2F transcription factor being validated. The oligoprecipitates will also be family, involved in cell cycle progression, analyzed by LC-MS/MS to identify new potential apoptosis, DNA damage repair, differentiation and E2F2 interacting proteins. development. Although classically E2F2 has been classified as a cell cycle activator, previous work in Innovative aspects our lab has demonstrated that it can also act as a • Selective enrichment of E2F2 containing cell cycle repressor (1). It is known that the protein complexes using an specific DNA transcriptional activity of E2F2 is dependent on its sequence as a bait. interacting partners (2), but the ones described so • Comparison between two different far are insufficient to understand the way E2F2 precipitations techniques for the identification regulates the cell cycle. Here we describe two of new E2F2 interacting proteins. different methods for the identification of new E2F2 interacting proteins. References (1) M. Murga et al, Mutation of E2F2 in mice Methods causes enhanced T lymphocyte E2F2 containing protein complexes were proliferation, leading to the development of immunoprecipitated (IP) with a specific antibody autoimmunity; Immunity 2001. against HA and oligoprecipitated (OP) using a (2) S. Schlisio et al, Interaction of YY1 with specific DNA (Chk1 promoter) containing the E2F E2Fs, mediated by RYBP, provides a consensus sequence as a bait, from HA-E2F2 and mechanism for specificity of E2F function; HA-E2F2 / HA-DP1 overexpressing cell lysates, EMBO J. 2002. respectively. Immunoprecipitations were ß- actin performed 5 times. In both cases the precipitates INPUT Chk1 were analyzed by western blot to confirm that the A B :OP antiHA precipitations were performed correctly. INPUT antiT E2F2 Components of the immunoprecipitated protein :IP complexes were identified by LC-MS/MS after tryptic digestion. E2F2 DP-1 IB: antiE2F2 IB: antiHA Results We have setup two different and complementary precipitation methods that allow us to enrich the Figure 1. (A) E2F2 was immunoprecipiated with anti-HA cell lysates in E2F2 and its interacting partners. but not with the control antiT antibody. (B) E2F2 and Starting from HA-E2F2 overexpressing 293T cell DP-1 were oligoprecipitated with the DNA containing the lysates, we were able to immunoprecipitate E2F2 E2F consensus sequence (Chk1) but not with the using a specific antibody against HA but not using control DNA (ß- actin). an unespecific antibody (Fig 1A). Moreover, A B 50 starting from HA-E2F2 and HA-DP1 45 Number of proteins 40 overexpressing 293T cell lysates, we were able to antiHA antiT 35 oligoprecipitate E2F2 and its dimerization partner 30 DP1, using a specific DNA (Chk1 promoter) 17 71 60 25 20 containing the E2F consensus sequence as a bait 15 10 but not with an unespecific DNA sequence (ß-actin 5 promoter) (Fig 1B). 0 We identified E2F2 and several potential E2F2 1 2 3 4 5 interacting partners by LC-MS/MS from the Number of replicates immunoprecipitates (Fig 2A). We consider specific Figure 2. (A) Number of proteins identified in 5 proteins those that only appeared in the problem immunoprecipitations against HA and 5 sample and in none of the negative control immunoprecipitations against SV40Ag T. (B) Number of replicates. Fig 2B shows the number of specific specific proteins present in one or more of the 5 proteins present in one or more of the 5 replicates replicates. performed. Quantitative phospho-proteomics elucidates phosphorylation-dependent transcriptional regulation of the yeast Mediator complex Gideon Oudgenoeg1, Joris Benschop2, Manuel Tzouros1, Tony Miles2, Frank Holstege2, Jeroen Krijgsveld1 1 Biomolecular Mass Spectrometry and Proteomics group, Utrecht University, Utrecht, the Netherlands 2 Genomics laboratory, University Medical Center Utrecht, Utrecht, the Netherlands Introduction application to the Mediator complex has linked Mediator is a 26-subunit protein complex that is kinase activity in the Mediator to transcriptional associated with RNA polymerase II. It is required regulation by this complex. The developed for transcription of all protein-coding genes in methodology will be applicable to other types of yeast and is functionally conserved from yeast to modifications, regulatory factors and organisms. mammals. One of the Mediator subunits is Cdk8, a protein kinase mainly involved in transcription Innovative aspects repression, notably through phosphorylation of the • Identification of kinase-specific repeated C-terminal domain of Rpb1 Pol II subunit. phosphorylation sites In addition, phosphorylation of other Mediator • Linking kinase activity to transcriptional subunits may serve as a mechanism of regulation by combination of genomics and transcription regulation. The aim of this study was proteomics. to identify Cdk8-dependent phosphorylation target sites within the Mediator complex. Methods Isolation of specific subunits for analysis of (1) Pinkse W. H. et al., Highly Robust, phosphorylation was performed by Tandem Affinity Automated, and Sensitive Online Purification (TAP), thereby co-purifying associating TiO2-Based Phosphoproteomics Applied subunits as well. Phosphorylated peptides in To Study Endogenous Phosphorylation in proteolytic digests of affinity-purified Mediator were Drosophila melanogaster; J. Proteome enriched by TiO2 affinity purification followed by Res., 7 (2), 687–697, 2008. nanoflow LC-MSMS (1) using a LTQ-FT mass spectrometer. Kinase- and condition-dependent (2) van de Peppel, J. et al., Mediator phosphorylation of Mediator subunits was inferred expression profiling epistasis reveals a from quantitative changes in phosphorylation using signal transduction pathway with wt and Cdk8 mutant strains, grown in normal and antagonistic submodules and highly 15N-labeled media, respectively. Targeted specific downstream targets. Mol. Cell 19, quantification of phosphorylated peptides of 511–522, 2005. interest was performed by Multiple Reaction Monitoring (MRM) using nano-RP-LC coupled to a hybrid triple quad - ion trap instrument. Results Multiple phosphorylation sites were identified in several Mediator subunits. Quantitative analysis revealed that several of these phosphorylation events were decreased in the Cdk8 mutant thus indicating that phosphorylation was Cdk8- dependent. To test functional relevance and putative regulatory role of candidate sites, point- mutants of Cdk8-dependent phospho-serines in Med15 were made. Expression profiles of these point-mutants were compared to wt, ∆Cdk8 and ∆Med15 deletion strains. This revealed that mRNA Figure 1.Modular organization of the Mediator Complex (2) and close functional relationships between profiles of Med15 point mutants were different from the Mediator modules and other transcriptional both wt and ∆Med15 deletion strains, but very regulators. similar to the ∆Cdk8 mutant. This indicates that Cdk8-mediated phosphorylation of Med15 is a direct modulator regulating gene transcription. The present work shows that quantitative phospho-proteomics is a powerful tool to reveal kinase-specific phosphorylation sites. Its Targeting Dynamic Changes of the CFTR Interactome in Cystic Fibrosis Sandra Pankow, Casimir Bamberger, William Balch, John R. Yates IIIrd Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Introduction between the different complexes and determine 70% of all cystic fibrosis cases are caused by the timely manner of the interactions. Eventually, deletion of phenylalanine 508 (∆F508) of the cystic close evaluation of these results might elucidate fibrosis transmembrane conductance regulator potential therapeutic targets for cystic fibrosis that (CFTR). In contrast to wild-type (wt) CFTR most of can be initially tested in the present system. the ∆F508 CFTR is incorrectly folded and consequently proteolytically degraded, probably due to loss of key interaction partners. To determine and distinguish differences in the Innovative aspects composition of protein complexes associated with • Development of a sensitive IP protocol for the endogenous wt and mutant CFTR during the endogenous membrane-embedded proteins lifecycle of the protein, we applied • Previously non-identified interaction partners immunoprecipitation and MudPIT (1) in a were determined for mutant and wt CFTR combined approach. • Dynamic changes in protein interactions during the protein lifecycle can be monitored Methods CFTR is a low abundant 12-transmembrane References protein that is hardly accessible by normal (1) M.P.Washburn et al., Large scale analysis experimental procedures. We therefore developed of the yeast proteome by multidimensional an immunoprecipitation (IP) protocol that provided protein identification technology; Nat. high sensitivity and allowed us to detect CFTR and Biotech 2001 associated protein complexes from patient-derived (2) X.Wang et al., HSP90 Cochaperone Aha 1 cell lines carrying either the ∆F508 CFTR gene or downregulation rescues misfolding of the re-inserted wt CFTR gene. Parental cells in CFTR in cystic fibrosis; Cell 2006 which the CFTR gene had been silenced were used as control. For better access to membrane- embedded proteins, membranes were slightly disrupted by applying gentle sonication before IP. Immunoprecipitated proteins were then further extracted with Methanol/Chloroform to remove interfering lipids. To possibly most extensively cover the network of interacting proteins, a modified MudPIT protocol capable of eluting hydrophobic peptides was applied. Results Using the newly developed strategy we were able to identify over 350 proteins that constitute directly or indirectly CFTR containing protein complexes. Of these, roughly 92% are newly identified as CFTR interaction partners and about 40% are specifically associated with the ∆F508 mutation. Whereas the previously reported network of CFTR interacting proteins (2) represents a core network allowing a relatively static view of the complexes associated with CFTR, the extended network enables us to follow the dynamic changes in the CFTR interactome that occur during folding and trafficking and that determine membrane stability, e.g. reflect the different cellular processes comprising the lifecycle of CFTR from nascence to degradation. Fractionation of the subcellular compartments will further allow to distinguish Cell cycle regulation of TBP complexes in human cells 1 2 1 2 W.W.M. Pim Pijnappel , Annemieke Kolkman , Marijke P.A. Baltissen , Albert J.R. Heck , and H. Th. Marc 1 Timmers 1 Department of Physiological Chemistry, University Medical Center – Utrecht, Universiteitsweg 100, 3584 CG 2 Utrecht, The Netherlands; Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands Introduction Genome wide studies have revealed waves of gene expression during the cell cycle. In addition, mitotic cells exhibit a transcriptional shut down. To investigate whether these events are regulated at the level of general transcription factors, we analyzed the compositions, abundances and post- translational modifications of TBP complexes during the human cell cycle using affinity purification and SILAC. Methods HeLa cells were transduced with flag-HA tagged TBP using retroviral gene delivery. Expression of tagged TBP matched endogenous expression. Cells were isotopically labelled using heavy arginine and lysine, and were grown in a 15 liter bioreactor. Comparisons were made between cells grown asynchronously and cells blocked in mitosis (using nocodazole) or in S phase (using thymidine). TBP complexes were affinity purified, digested by trypsin and analyzed by nano-LC- LTQ-FT-MS. Phosphopeptides were enriched using titanium oxide precolumns. Results Our data indicate that the compositions of stable TBP complexes remained largely intact, whereas their relative abundances changed during the cell cycle. In addition, we detected a number of novel phosphorylations on TBP-associated factors (TAFs), some of which were enriched in mitosis or in S phase. The function of one of the mitotic phosphorylations has been studied in more detail using a functional replacement with phosphopreventing or phosphomimetic mutant proteins and chromatin association and transcription assays. The results of these analyses will be presented. Innovative aspects • Generic double affinity purification method for medium abundant transcription factor complexes in human cells • Adaptation of SILAC methodology and cell cycle block and release for large scale bioreactor-based human cell culture • In-depth characterization of cellular changes in human protein complexes using state of the art mass spectrometry and biological validation. Examination of the protein complexes bound on gal operon promoter of Thermoanaerobacter tengcongensis 1,2 1, 2 3 1,2 Zhong Qian , Fan Wei , Li Guo , Siqi Liu 1 2 3 Beijing Genomics Institute, CAS, Beijing, China; Beijing Proteomics Institute, Beijing, China; Institute of Microbiology, CAS, Beijing, China Introduction (TTE0779) in 2U have not been reported to be T. tengcongensis is an anaerobic, thermophilic related with DNA binding as well as transcriptional eubacterium. Its genomic and proteomic profiles regulation. Further experiments are being carried were well investigated. In our laboratory, a novel on to separate these complexes using gal operon was discovered from this bacterium, immunoprecipitation approach for functional which is composed of five genes, TTE1925, analysis. TTE1926, TTE1927, TTE1928, and TTE1929, and induced its gene expression by galactose. The T. Innovative aspects tengcongensis gal operon is likely to have two • We used the EMSA-LC MS/MS to detect the promoters, O1 and O2, however, there is lack of unknown regulation protein complexes in the information how the regulatory proteins interact regulation mechanism investigation for the with these DNA regions. Therefore, we initiated the thermophilic bacterium. study to explore the proteins potentially bound to the promoters. References (1) D. Stenger, et al, Mass spectrometric Methods identification of RNA binding proteins from T. tengcongensis cells were lysated in the buffer dried EMSA gels; J. Proteome Res. 2004, containing lysozyme on ice for 4h. Synthesized 3(3): 662-664. 32 ssDNA probes were annealed, labeled with [γ- P] and mixed with the lysates. After incubation for 15m, DNA-protein complexes were separated through 8% native gel. According to the EMSA results, the autoradiographic shifted bands were obviously observed either in galactose or glucose medium, implying some proteins did interact with the probes. To detect the components in the shifted DNA-protein complexes, we excised the four shifted bands, termed as 1A, 2A, 1U and 2U, and identified the protein complexes by LC MS/MS after completely tryptic digestion (1). For data analysis of LC MS/MS results, a stringent criterion was employed, an identified protein confirmed from 3 unique peptides at least. Results Figure. EMSA analysis of the DNA-protein complexes. Totally we have identified more than 30 proteins This figure indicates the obvious shifted bands, implying for each complex. Since the excised bands might some proteins have an interaction with the probes contain some proteins that were non-specifically compared to the controls (O1 and O2). bound to DNA or the background contamination, the redundant identifications overlapped among these bands were removed in the list of DNA- protein complexes. After the treatment, 3 proteins were identified in complex 1A, 1 in complex 2A, 1 in complex 1U and 4 in complex 2U. In glucose medium, 2 important proteins were identified, HPr and PEP-protein kinase. These two proteins were reported to be involved in a classical carbon metabolism regulation pathway, carbon catabolite repression (CCR). However, in galactose medium, TTE1926 (transcriptional regulator) was identified as dominant protein bound to 1A and 2A. TTE1926 is a component of T. tengcongensis gal operon and is expected to possess a DNA binding structure. ATP:corrinoid adenosyltransferase in 1A, acetate kinase and hypothetical protein Background proteins in immunoprecipitations: what are they and how reduce them Patrice Waridel, Bastienne Jaccard, Alexandra Potts and Manfredo Quadroni Protein Analysis Facility, Center for Integrative Genomics, University of Lausanne, Switzerland Introduction Immunoprecipitation (IP) of a protein of interest is frequently used to identify potential interaction Innovative aspects partners in the cell. The ubiquitous presence of variable levels of unspecific "background" proteins, • Analysis of immunoprecipitation background however, introduces noise in the data and often proteins on a collection of samples results in both false positives and false negatives. • Identify critical steps and reagents We have analyzed the composition of this protein • Improved protocol with lower background background through a survey of a number of mock IPs prepared in different labs. Also, in a parallel study under controlled conditions we have tried to References determine the origins of the background and to modify protocols to reduce it. None Methods Exact procedures for the IPs of "survey" samples varied from lab to lab. All IPs were carried out from human cell extracts, using antibody-based affinity steps. Proteins bound to beads after IP were eluted by boiling in SDS and fractionated on mini SDS-PAGE gels. Molecular weight regions were cut and in-gel digested. Peptide mixtures were analysed by LC-MS/MS on a Thermo LTQ- Orbitrap. For the protocol optimization study, cell lysis and IP were performed with a 1% NP-40, 150 mM NaCl buffer and with ProteinA-Sepharose beads. A human IgG fraction and an anti-PCNA monoclonal antibody were used for the tests. Several IP parameters were varied for optimization. Analysis was performed by gel and fluorescence staining as well as digestion and LC- MS/MS as described above. Results Comparative analysis of 10 Ab-based mock (negative control) IPs prepared under different conditions highlighted the recurrent recovery of Figure 1. Sypro ruby stained gel of IPs performed on several families of proteins. From these data, a set HeLa cell extracts with 5 μg human IgG using our of about 130 proteins was identified, which were modified protocol vs. a standard one. Bands stronger in recovered in at least 80% of all experiments and the standard protocol are indicated by blue circles. The are thus very likely nonspecific background. lane on the right contains only IgGs (starting material). We have then carried out tests under controlled conditions using a commonly used IP protocol which employs ProteinA-Sepharose beads, by subtracting one component at the time. The results indicate that most background proteins are recovered because of interaction with the Sepharose beads. Furthermore, aggregation and precipitation phenomena occurring in the 1-2 hours after lysate preparation are responsible for a large part of the background. We show that treatment with benzonase coupled with an additional centrifugation step one hour after lysis and before addition of the beads are simple and effective ways to strongly reduce nonspecific background. OPTIMIZED IMMUNOPRECIPITATION AND CO-IMMUNOPRECIPITATION USING DYNABEADS Immunoprecipitation and co-immunoprecipitation are classical methods used to isolate specific proteins or protein complexes from biological samples. Methods use the antibody-antigen reaction principle to identify a protein that reacts specifically with an antibody in a mixture of proteins so that its quantity, physical characteristics or interaction partners can be determined. Traditionally, sepharose and agarose slurries have been used, but more recently magnetic beads have gained popularity due to shorter and simpler protocols. Magnetic Dynabeads are ideal for immunoprecipitation. The rapid procedure permits the isolation of labile composites that might otherwise dissociate during long incubation times (or be damaged by proteases), there is no upper size limit for the complex to be isolated (ideal for complex pull-down) and the surface properties give very low non-specific binding. With Dynabeads there is no fear of losing beads (as with spun down resin) and you can scale down the procedure to reduce the consumption of expensive antibodies. Here we show data of improved immunoprecipitation and co-immunoprecipitation protocols, and demonstrate the usefulness of combining GFP-tagged fusion proteins with Dynabeads to enable the combined visualization and pull-down of proteins. Immunoprecipitation Your captured primary antibody can be cross-linked to the Dynabeads® or used directly for affinity purification of pure target protein, peptides, protein complexes or other antigens (fig. 2). The benefits of magnetic protein separation are reaped in a wide variety of applications, as documented in numerous publications. For details, protocols and references, please refer to: www.invitrogen.com/dynal Dynabeads® magnetic separation technology is also easy, rapid and efficient for small-scale magnetic purification of antibodies. Within minutes, you can capture pure and highly concentrated antibodies directly from samples such as saliva, ascites, serum and tissue culture or hybridoma supernatants. have several limitations compared to magnetic beads which and more . In this study we show data on improved protocols for immunoprecipitation using Dynabeads and compare the performance with other technologies. This can be agarose / sepharose beads (slurries), but more recently Protein A or -G conjugated magnetic beads have gained popularity[citation needed] due to shorter, simpler protocols and less non- specific binding Proteasome Diversity Analysed by Isotope Labelling and Protein Quantitation 1 2 2 2 Reinout Raijmakers , Celia R. Berkers , Annemieke de Jong , Huib Ovaa , 1 1 Shabaz Mohammed and Albert J.R. Heck 1 Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, the Netherlands 2 Division of Cellular Biochemistry, Tumor Biology, and Immunology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Introduction direction (2). In addition, we found also the PA28 The proteasome is the protein degradation cap proteins to be much more abundant in the machinery of all eukaryotic organisms and spleen proteasome. As this particular cap structure archaebacteria. It degrades protein to short is known to associate mainly with the immuno- peptides of about seven to eight amino acids, proteasome, this finding corresponds nicely with which can be further processed to single amino the upregulation of the immunoproteasome beta acids to be reused by the cell in the synthesis of subunits in that tissue. new proteins. Inhibitors of the proteasome are strong inducers of apoptosis and can be used as Innovative aspects anti-cancer therapeutics. To enhance therapeutic applications targeting the proteasome, it is vital to • Quantitation of proteasome complexes from understand in great the detail the diversity of cow tissue samples reveals tissue specific proteasome complexes in various tissues. composition of the proteasome Methods References To analyze proteasome diversity, we first purified 20S proteasome core complexes from cow liver 1. Hsu, J. L., S. Y. Huang, N. H. Chow, and S. H. and spleen tissue. Proteasome purification was Chen. 2003. Stable-isotope dimethyl labeling for achieved by sequential ammoniumsulphate quantitative proteomics. Anal Chem 75:6843-52.2 precipitation, sucrose gradient centrifugation and 2. Berkers, C. R., F. W. van Leeuwen, T. A. Groothuis, V. Peperzak, E. W. van Tilburg, J. weak anion exchange chromatography. Following Borst, J. J. Neefjes, and H. Ovaa. 2007. Profiling digestion, the two proteasome samples were then proteasome activity in tissue with fluorescent labelled using an improved version of chemical probes. Mol Pharm 4:739-48 labelling of protein digests by dimethylation of peptide N-termini and lysine side chains. This is achieved with a mixture of formaldehyde and cyanoborohydride, using either normal or deuterated formaldehyde (1). Labelled samples are analyzed by LC-MS/MS and quantitative differences between the samples are determined by comparing the intensity of the light and heavy labelled peptides as determined by the mass spectrometer. Results After purification and LC/MS analysis we were able Figure 1. Quantitation of proteasome subunits in cow to identify all 14 subunits (7 alpha and 7 beta spleen and liver tissue. Proteasome subunits are sorted subunits) of the core of the proteasome complex, from more abundant in the liver proteasome (left side, as well as the three alternative beta subunits which purple area) to more prominent in the spleen are only present in a specific subclass of the proteasome (right side, pink area. See the legend for the different classes of proteasome subunits included in the proteasome, called the "immunoproteasome". In graph. addition, we identified two proteins belonging to the PA28 regulatory cap, which is one of the cap structures that can regulate proteasome activity. Differential quantitation of all of these proteins between the proteasomes purified from cow liver and spleen revealed distinct differences between the complex in these tissues (Figure 1). One of the catalytic beta subunits of the proteasome was significantly more abundant in the liver, whereas the three beta subunits of the immunoproteasome were more present in the spleen sample, corresponding to previous indications in that Protein complex studies of mouse brain using a new proteomics platform 1 1 1, 2 Ram al i n gam R aj k um ar , Chen-Chung Liao , a nd Yeou-Guang Tsay 1 2 Proteomics Research Center and Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan extract. Two major parameters were recorded with Introduction each identified protein, the molecular masses of its Protein complex formation is one of the major primary and quaternary structures. These items of events that regulate the protein functions (Collins information were instrumental in establishment of et al., 2007, Wang and Huang, 2008). Therefore, the protein complex map. Functional comprehensive mapping of protein-protein categorization of proteins in GFC fractions shows interactions within the proteome will inevitably that the proteins for certain functions have a become a challenging mandate in proteome special tendency to become macromolecular research (Andrew et al., 1999). In the present structures in the brain tissue. Thus, protein investigation, we report the application of a new complex mapping analysis may help us to proteomics platform to the studies of protein understand the significance of protein complex complexes in the entire mouse brain proteome. assembly/disassembly in the overall function of cells or tissues. Methods References Seven weeks old C57/B16 mice were maintained 1. Andrew J.Link et al, Direct analysis of protein under standard breeding condition. The brains complexes using mass spectrometry; Nature. were removed after decapitation and immediately 1999, 17; 676-682. frozen on dry ice, minced and homogenized in ice- 2. Collins, S. R., et al., Toward a comprehensive cold extraction buffer. The homogenate was atlas of the physical interactome of centrifuged and the supernatant was fractionated Saccharomyces cerevisiae; Mol. Cell. with gel filtration chromatography (GFC) and the Proteomics. 2007, 6; 439-450. proteins from each GFC fraction were resolved 3. Xiaorong Wang and Lan Huang Identifying with SDS-PAGE. All of the protein bands in the dynamic interactors of protein complexes by gels were digested with trypsin and then analyzed quantitative mass spectrometry; Mol. Cell. with liquid chromatography-tandem mass Proteomics. 2008, 7; 46-57. spectrometry (LC-MS/MS). Turbo SEQUEST algorithm and our in-house computer software were used to interpret the LC-MS/MS data, and to construct the protein complex map. Results With this method, we identified more than 1,500 different proteins from the mouse brain sample. Based on the protein staining intensities, we estimated that ~ 60% of proteins were present as multimeric protein structures in our brain tissue LC-MS Analysis to Identify Ryanodine Receptor 1 (RyR1) Protein-Protein Interactions 1 1 4 3 1,* Tim Ryan , Parveen Sharma , Alex Ignatchenko , David H. MacLennan , Anthony O. Gramolini and Thomas 2,4,* Kislinger 1 2 3 Departments of Physiology, Medical Biophysics, Banting and Best Department of Medical Research, 4 University of Toronto, Ontario Cancer Institute, Division of Cancer Genomics and Proteomics Toronto, ON, Canada * Equal senior authors Introduction immunoprecipitation assays. This protein-protein Excitation-contraction coupling is defined as the interaction analysis provides a framework for a process linking action potential to contraction in more detailed analysis of RyR1 function. striated muscle. This process depends on a large macromolecular protein complex, the central 2+ element of which is the Ca release channel, Innovative aspects ryanodine receptor 1 (RyR1) (1). It has been • To date, there has been no large scale protein- demonstrated that protein binding partners protein interaction analysis of RyR1. regulate RyR1 via its cytosolic domain and • We have identified novel RyR1 interacting mutations in RyR1 and its interacting proteins proteins. result in disease. Here, we describe the purification and LC-MS analysis of affinity-tagged RyR1 protein complexes with the overall aim of References understanding calcium ion movement at a (1) Du, G.G., et al., Topology of the Ca2+ molecular level. release channel of skeletal muscle sarcoplasmic reticulum (RyR1). Proc Natl Acad Sci U S A, 2002. 99(26): p. 16725- Methods 30. Full length RyR1 rabbit cDNA and a truncated, cytosolic RyR1 construct were affinity-tagged with 6x-histidine and streptavidin binding peptide (SBP). These constructs were introduced into HEK293 cells and C2C12 mouse skeletal myocytes enabling column purification of RyR1 protein complexes under non-denaturing conditions. The purified tagged proteins were subjected to immunoblotting experiments to verify stable expression and purification of clones. The purified complexes were analyzed using 1- dimensional gels and by gel-free LC-MS. A filtering algorithm was applied to all putative results to obtain a measure of the statistical reliability (confidence score) for each candidate identified Figure 1. Histidine-tagged RyR1 purified from and only proteins with confidence value ≥ 99% and transfected C2C12 muscle cells using Ni-NTA identified with ≥ 2 peptides were accepted. chromatography. Eluted protein complexes were immunoblotted and probed with an anti-RyR1 antibody. Results Here, we present data using tandem affinity purification tagged (streptavidin-binding, and HIS) proteins transfected into HEK293 and C2C12 muscle cells, followed by gel-free LC-MS to identify protein-protein interactions in skeletal muscle. Column purification of tagged proteins from transfected HEK and C2C12 shows stable expression and purification of clones by immunoblotting experiments. Figure 1 shows ryanodine receptor 1 (RyR1) from C2C12 myocytes, purified on a Ni-NTA column under non- denaturing conditions. LC-MS analysis of the purified protein complex showed that we have identified 5 novel RyR1 interacting proteins and we are currently validating these interactions using conventional co-expression and Quantitative 20S proteasome analysis from 5FU-induced apoptotic Jurkat T cells by SILAC, SDS-PAGE, and 2DE based analysis Frank Schmidt, Alexander Kloos, Martin von Bergen, Burkhardt Dahlmann, Peter Jungblut, and Bernd Thiede Department of Proteomics, Helmholtz Institute, Permoserstr. 15, 04318 Leipzig, Germany Introduction Innovative aspects DNA damage and subsequent induction of • Combination of SILAC and 2-DE apoptosis is possibly the primary cytotoxic mechanism induced by DNA-binding anticancer drugs. Apoptosis is a form of programmed cell death and can be triggered in a cell through either the extrinsic or the intrinsic pathway. In this study, quantitative 20S proteasome analysis of 5-fluoro- uracil-induced apoptosis of Jurkat T cell lysates was performed in order to identify differentially regulated proteasomal subunits. Methods Proteasomal proteins were labelled in cell culture with stable isotopes of arginines and lysines, fractionated by SDS-PAGE and 2-DE gels and further analyzed by MALDI-TOF/TOF-MS and LC- orbitrap MS. In a first step, cell lysate was subjected to a gel filtration on a Sepharose 6B column, in order purify the proteasome. Afterwards, the proteasome fraction was subjected to 1D PAGE and bands were digested by trypsin and identified as well as quantified by LC-orbitrap MS. In addition, a more detailed analysis was performed by 2-DE gel analysis where the protein species were cut out and digested with trypsin. PMF was achieved by MALDI-TOF/TOF-MS in order to identify and quantify proteasome subunits. Results In total, 26 bands were digested with trypsin and further analyzed by LC-orbitrap MS. Beside non- related proteasomal proteins, all α- and β-subunits were identified within at least one band excluding the immunosubunits β-1i, β-2i, and β-5i. With exception of α-7 (also known as C8, α7_sc, PSA3), all subunits showed an equally ratio of 1:1 using the average ratios belong to all peptides of a subunit. In case of α-4, α-7, β-4 and β-7 subunits were detected in two different bands with different molecular weights. Here, 20S proteasome species of α-4, β-4, and β-6 showed an equally ratio of 1:1 in contrast to the species of α-7, where the high molecular one was clearly down regulated during apoptosis (0.64) compared to the low molecular one (1.06). In order to explain the different ratios between the α-7 subunits, a 2-DE gel was performed. The ratio of low molecular α-7 subunit showed the same down-regulation as in the 1D PAGE. The detailed analysis of the MS spectra yields to a specific methylation only present in the regulated α-7 species. Proteomic Analysis of Membrane Protein Complexes: Lessons from Affinity-Purified Ion Channels and Receptors § §# # # Uwe Schulte , Catrin S. Müller , Wolfgang Bildl and Bernd Fakler § ) Logopharm GmbH, Schlossstr. 14, 79232 March-Buchheim, Germany # ) Institute of Physiology, University of Freiburg, Hermann-Herder-Straße 7, 79104 Freiburg, Germany Introduction examples provide a general basis for studying Membrane proteins represent a major challenge in protein networks at the plasma membrane and proteomics, especially in protein-protein interaction may serve to establish increased quality standards studies. Currently, affinity purification coupled to for affinity-based proteomics. LC-MS/MS sequencing is regarded as the method of choice. Although successful in some cases, this Innovative aspects approach has multiple pitfalls and lacks general • Large-scale identification of signaling quality standards. The most critical factors, namely supercomplexes at the plasma membrane protein solubilization, antibody-related effects and • Native-source based proteomics with highest unbiased MS data evaluation are addressed based sensitivity and reliability on more than 1000 affinity purifications of ion • Characterization of previously unapproachable channels and receptors performed and analyzed or functionally unassigned proteins by our group. References Methods (1) Berkefeld H, Sailer CA, Bildl W, Rohde V, Thumfart Membrane fractions were prepared by JO, Eble S, Klugbauer N, Reisinger E, conventional methods from native tissues and Bischofberger J, Oliver D, Knaus HG, Schulte U, subjected to solubilization assays. A selection of Fakler B: BKCa-Cav channel complexes mediate 2+ + standardized non-denaturing detergent buffers rapid and localized Ca -activated K signaling (ComplexioLytes) was tested for efficiency and (2006). Science 314: 615-20 complex integrity. Solubilized protein complexes (2) Schulte U: Protein-protein interactions and subunit composition of ion channels (2008). CNS Neurol were affinity-purified using immobilized antibodies. Disord Drug Targets 7: in press Pre-immune IgGs or target knockout preparations served as negative controls. Isolated proteins were resolved by SDS-PAGE, silver-stained and in-gel digested with trypsin. Extracted peptide mixtures were finally resolved on an UltiMate 3000 nano- HPLC reverse-phase system directly coupled to a linear ion trap ICR hybrid mass spectrometer (LTQ-FT). Proteins were identified using Mascot; LC-MS/MS data was quantitatively evaluated with proprietary software. Results Comprehensive identification of ion channel and receptor-associated proteins requires a combination of technical optimization, appropriate controls and quantitative evaluation. Optimized solubilization with ComplexioLytes shows more than 80% efficiency while preserving high molecular weight target complexes of 0.5-2.5 MDa. Antibody quality is of critical importance: unpredictable cross reactivities, biased selection of target complex populations and competition with associated proteins are common sources for artefacts. These problems can by resolved by using multiple antibodies, different purification conditions and target knockouts as stringent controls. Finally, relative quantification by LC- MS/MS is required for validation of protein specificity. Application of this technology has led to Figure 1. Proteomic analysis of BK channel complexes the identification of novel and functionally relevant as an example. Optimization of critical steps and careful interaction partners of a number of ion channels quality controls resulted in high BKα sequence coverage (1, 2) and G-protein coupled receptors. These and identification of several novel interaction partners. Analysis of subunit composition of the 20S proteasome of Plasmodium falciparum 1,2 1 2 2 2 1 1 N. Sessler , W. Schütz , R. Fendel , B. Mordmüller , P. G. Kremsner , T. Lamkemeyer , A. Nordheim 1 Proteome Center Tübingen, Interfaculty Institute of Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany 2 Department of Parasitology, University of Tübingen, Wilhelmstraße 27, 72074 Tübingen, Germany Introduction This analysis revealed at least three variants of the Malaria due to Plasmodium falciparum causes proteasome complexes at ~700 kDa (a, b, c; see more than 1 million deaths per year. Symptoms fig. 1B). occur during the erythrocytic phase of replication, We excised three areas in the gel containing the characterized by four stages: merozoites, ring- proteasome complexes from schizont stage stage trophozoites (rings), trophozoites and lysates as determined by Western blotting and schizonts. The 20S proteasome of P. falciparum is analysed the protein composition by a classical 2D essential for differentiation and replication, and is a PAGE (see fig. 2) All gels of the three excised gel promising therapeutic target. slices displayed a comparable spot pattern in the In order to gain a more detailed knowledge about expected mass range of 20 to 35kDa. We structure, function and modifications of the observed differences in spot intensity between the proteasome during the life cycle of this parasite we 2D gels which correlates with the decreasing analysed the proteasome by multi-dimensional intensities of the proteasome complexes seen in electrophoresis and mass spectrometry. BN-PAGE (not shown). We analysed the corresponding proteins of complex a (see fig. 2) by Methods LC-MS/MS and identified 13 proteasome subunits. Parasites (P. falciparum strain 3D7A) were cultured and synchronised using standard pH 3 2. dim: IEF pH 10 [1] methods. At a parasitaemia of approx. 5%, cells of the respective stage were harvested after 220 removal of contaminating erythrocytes. [2] 116 3. dim: SDS-PAGE For three-dimensional analysis two sets of total protein extracts were separated by blue-native 66 [3] 55,6 polyacrylamide gel electrophoresis (BN-PAGE) . After electrophoresis one set was analysed by Western blot to detect the proteasome complexes 36,5 7 using an antibody against the 20S proteasome. By 4 8 29 1 5 matching the blot to the second set on the non- 9 12 2 3 6 10 13 blotted gel the corresponding areas to be excised were determined. The proteins were eluted and 20 11 subsequently analysed by classical 2-dimensional (2D) PAGE followed by nano-liquid chromato- Fig. 2: Classical 2D-gel of the eluted proteins from the graphy and tandem mass spectrometry (LC- proteasome complex a of schizonts (see fig. 1A, lanes 3 + MS/MS) analysis. 1B). The 13 marked spots were analysed by LC-MS/MS. Results To date we have analysed the 20S proteasome in Similar analyses of the other erythrocytic phases three of the four blood stages of P. falciparum of replication are ongoing. (rings, trophozoites, and schizonts). Innovative aspects A 1 2 3 B Fig. 1: Determination • Separation of total protein extracts of of the areas in the gel P. falciparum and detection of three containing proteasome proteasome complexes by BN-PAGE. 720 c complexes. • Direct identification of the proteasome subunits b A: Western blot of BN- a using three-dimensional electrophoresis. PAGE of proteins of rings (1), trophozoites 480 References (2) and schizonts (3). 1. dim: BN-PAGE B: BN-gel of schizonts [1] Mordmüller B. et al., Molecular & Biochemical proteins, showing Parasitology (2006), 148, 79–85. 242 proteasome [2] Werhahn W. and Braun H.-P., Electrophoresis complexes marked in (2002), 23, 640-646. red (a, b, c). [3] Schägger H., Cramer W.A., von Jagow G., 146 Analytical Biochemistry (1994), 217, 220-230. Comparative mass spectrometry of the endogenously expressed nuclear and cytoplasmic exosome Silvia A. Synowsky, Reinout Raijmakers and Albert J. R. Heck Biomolecular Mass Spectrometry and Proteomics group, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands Introduction Proteins are organized in large RNA binding domain of Csl4, is significantly protein complexes that form an extensive network upregulated in the nuclear exosome. in the cell [1]. Since these protein assemblies are essential for most cellular functions, it is of high Innovative aspects importance to determine their constituents, • Combination of TAP with macromolecular topology and subtle differences especially between mass spectrometry and quantitative related protein complexes. Here we employ a proteomics reveal structural features and multiplexed MS approach to investigate subtle changes in endogenously expressed endogenously expressed protein complexes by heterogeneous protein complexes native MS and quantitative proteomics. Our • Macromolecular MS/(MS) identifies subunit strategy is applied on the yeast nuclear and composition, the most common stable core of cytoplasmic exosome to identify subcomplexes exosomes and gas-phase dissociation and to quantitate differences in their patterns phosphorylation status. • Quantitative proteomics show relative differences in the phosphorylation status of the Methods The exosomes were purified using a nuclear and cytoplasmic exosome nuclear and cytoplasmic specific TAP tagged protein (Rrp6 and Ski7) [1]. The intact exosomes References were first analyzed in the MS and MS/MS mode [1] Gavin AC, et al., Functional organization of using a modified QToF1 instrument [2]. Typical the yeast proteome by systematic analysis of settings were a capillary voltage of 1400 V and a protein complexes. Nature. 2002; 10(415): sample cone voltage of 150 V. In the MS/MS mode p.141-7. the collision voltage was raised stepwise from 50 [2] van den Heuvel RH, et al., Improving the to 175 V to observe dissociation patterns of the performance of a quadrupole time-of-flight selected precursor. Quantitative information about instrument for macromolecular mass the nuclear and cytoplasmic exosome proteins and spectrometry. Anal Chem. 2006; 78(21): their phosphorylation status were obtained by p.7473-83. tryptic in-gel digestion followed by dimethyl [3] Synowsky SA, et al., Probing genuine strong labeling with heavy and light formaldehyde interactions and post-translational respectively. The peptides were mixed and modifications in the heterogeneous yeast analyzed using an LTQ-FT. Prior to MS analysis exosome protein complex. Mol Cell the phosphopeptides were enriched using TiO2. Proteomics. 2006; 5(9): p. 1581-92 Results The nuclear and cytoplasmic exosome complexes exhibit the same core complex in native MS. The mass corresponds to the hexameric ring plus Dis3, Rrp4 and Rrp40. Therefore the 40+ charge state of the nuclear variant has been subjected to gas-phase dissociation in MS/MS. Surprisingly at low collisional voltage Rrp40 is ejected with few charges from the precursor indicating a native fold upon dissociation. At further increasing collision voltage a second dissociation pathway emerges in which partially unfolded Rrp4 dissociates from the precursor. Quantitative proteomics identifies all common proteins between the nuclear and cytoplasmic exosome to be unchanged. Differences were observed in strictly nuclear proteins (Rrp6, importin / , Lrp1 and Ynr024w) and the cytoplasmic protein Ski7. As shown previously several exosomal proteins contain at least one phosphorylationsite [3]. Here we could relatively quantify several of these phosphorylationsites between the nuclear and cytoplasmic variant. Most importantly we could establish that the P-site at serine 94, located in the Identification of intracellular molecules interacting with the stress-induced, psoriasis susceptibility-related non-coding RNA, PRINS 1 2 3 1,4 Krisztina Szegedi , Mária Antal , István Németh , Zsuzsanna Bata-Csörgő , 1,4 1,4 4 Attila Dobozy , Lajos Kemény , Márta Széll Department of Dermatology and Allergology, University of Szeged, Korányi fasor 6, 6720 Szeged, Hungary Introduction when cells are released from cell quiescence, Previously we have identified a non-coding RNA while their protein level is highest in differentiating gene, PRINS (Psoriasis susceptibility-related RNA cells. gene Induced by Stress) that is expressed at an We hypothesize that the non-coding PRINS RNA elevated level in psoriatic uninvolved epidermis may be a structural element of a molecular compared to healthy epidermis and in cells under complex playing a role in stress-induced cellular various stress conditions. Those expression data processes and the abnormal functioning of this suggests a role for PRINS in psoriasis complex may contribute to the pathogenesis of susceptibility and in cellular stress response (1). psoriasis. Our aim was to access the cellular function of PRINS by identifying intracellular molecules interacting with this stress-induced non-coding RNA. Innovative aspects • The non-coding RNA, PRINS physically interacts with chaperon molecules Methods nucleophosmin and GRP94 in HaCaT We performed in vitro experiments with a keratinocytes. PRINS RNA may be a structural ribonucleoprotein purification kit. As a target element of a molecular complex including template, we used a transcript containing the nucleophosmin or/and GRP94 19mer sequence that has previously effectively • The expression of both nucleophosmin and knocked down the expression of PRINS both in GRP94 depends on proliferation/differentiation HeLa and HaCaT cells. We hypothesized that this state of syncronised HaCaT cells, thus the region may play an active role in binding other posttranscriptional regulation might play an ribonucleic acids and/or proteins. With the help of important role in their protein expression Matrix-Assisted Laser-Desorption Ionization Time- • Here we provide the first data that of-Flight (MALDI-TOF) method we identified two nucleophosmin might serve as a marker of proteins in HaCaT cell lysates that physically higher proliferation rate in the basal layers of interact with PRINS RNA. To further characterize psoriatic involved epidermis the identified proteins, we used immunohistochemistry, RT-PCR and Western blot References techniques to detect their mRNA and protein (1) E.Sonkoly et al, Identification and expression in HaCaT cell cultures and in healthy characterization of a novel, psoriasis and psoriatic skin samples and other various susceptibility-related noncoding RNA tissue samples. gene, PRINS; J. Biol Chem 2005 Jun 24; 280(25):24159-67 (2) V. Palaniswamy et al, Nucleophosmin is Results selectively deposited on mRNA during We identified two proteins in HaCaT cell lysates polyadenylation.Nat Struct Mol Biol. 2006 that physically interact with PRINS RNA. One of May;13(5):429-35 the identified proteins, nucleophosmin is a (3) P. Fortugno et al, Regulation of survivin ubiquitously expressed nucleolar phosphoprotein function by Hsp90; Proc Natl Acad Sci U S which shuttles continuously between the nucleus A. 2003 Nov 25;100(24):13791-6 and the cytoplasm (2). The other PRINS RNA binding protein, GRP94 is a molecular chaperone heat shock protein (3). Immunohistochemical experiments revealed that there is no difference in the expression of GRP94 protein when compared among healthy, psoriatic uninvolved and involved skin samples, while the expression of nucleophosmin is significantly elevated in the dividing cells of the basal layer. In vitro experiments performed on synchronised HaCaT cells revealed that the expression of both nucleophosmin and GRP94 mRNA is highest A Quantitative Proteomics Approach to Reveal Dynamics in Transcription Factor Complexes H.Th.M. Timmers*, F. Mousson*, P. de Graaf*, A. Kolkman#, W.W.M. Pijnappel*, B. Gevers^, A.B. Houtsmuller^ and A.J. Heck# *Department of Physiological Chemistry, UMC-U, Universiteitsweg 100, 3584 CG Utrecht; # Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands and ^Department of Pathology, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, Netherlands. The present methods to study the dynamics of spectrometric analysis. We used a standard multi-protein complexes are limited. We have combination of affinity purification and stable developed a quantitative proteomics strategy to isotope labeling (SILAC) to discriminate specific discriminate stable from dynamic subunits in from non-specific TBP interactors. By this protein complexes. This strategy is generally approach we would classify the known interactor, applicable and was used to examine the dynamics BTAF1, as non-specific. Thus, we compared of TBP transcription complexes. different affinity purification protocols and showed that a combination of different protocols can reveal Transcription in eukaryotic cells requires the information on the specificity and dynamics of continuous assembly and disassembly of the protein complexes. We find that the dynamic transcription machinery on gene promoters. The behaviour of BTAF1 is inhibited in mitosis and that association of the TFIID complex to gene this property is not shared by the TFIID, SL1 and promoters is the first step in assembly of RNA TFIIIB complexes. polymerase II transcription complex. The TATA- binding protein (TBP) can stably interact with TBP- Independent support for our quantitative associated factors (TAFs) to form (at least) four proteomics strategy comes from in vivo cell different TBP-TAF complexes (SL1, TFIID, B- imaging experiments. To measure TBP-complex TFIID, and TFIIIB) in the eukaryotic nucleus. While mobility in living cells we performed FRAP comparison of TBP-binding surfaces for TAF (fluorescence-recovery-after-photobleaching) subunits of these complexes explains their experiments using GFP-TBP expressing human mutually-exclusive binding to TBP, little is known cells. In this, the majority of TBP (~70%) displays a about stability of TBP-complexes and exchange mobile behavior, which is not an intrinsic properties of TBP and TAFs. To address this we biochemical property of TBP. Interestingly, the applied a SILAC-based proteomics strategy to mobile fraction of TBP molecules is decreased analyze subunit exchange in human cell extracts. after knockdown of BTAF1 expression. In contrast, We find that the BTAF1/hMot1 subunit of B-TFIID overexpressed BTAF1 increased the mobility of exchanges readily, which contrasts with the TBP molecules in vivo. behavior of other TAFs. Together we provide a generic quantitative These results were obtained by first generating a proteomics strategy to investigate regulation of the human cell line expressing a tagged version of dynamics in multi-protein complexes. This TBP. This allows affinity purification of the four revealed that the BTAF1 protein regulates the TBP-TAF complexes as shown by mass dynamics of the transcription factor TBP. Proteomic Analysis of Ubxd1: Identification of Novel Interaction Partners 1 2 2 1 Julian Vasilescu , Daniel Zweitzig , Dale S. Haines , and Daniel Figeys 1 Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, CANADA 2 Fels Institute for Cancer Research, Temple University, Philadelphia, USA Introduction Innovative aspects Ubxd1 is an adapter protein that was recently • First interaction mapping study of Ubxd1 identified as a p53 regulator that plays a role in cell • Novel Ubxd1 interaction partners identified cycle arrest and apoptosis (1). For this study, we • In-house software application created to performed a series of targeted protein interaction analyze multiple interaction datasets mapping experiments (2) in a human cancer cell line to identify novel interaction partners of Ubxd1. References Co-immunoprecipitation and immunofluorescence (1) Zweitzig et al, The ubiquitin selective experiments were then performed to validate a chaperone p97 and adapter UBXD1 number of these interactions. promote HAUSP-mediated MDM2 deubiquitination and suppression of p53 Methods function; (submitted) 2008. Human H1299 cells were transfected with (2) Vasilescu et al, Mapping protein-protein constructs encoding Ubxd1 with a carboxy- interactions by mass spectrometry; Curr terminal Flag- or HA- epitope tag. Cells were Opin Biotechnol; 2006. harvested and lysates were subjected to immunopurification using anti-Flag or -HA antibodies. Purified samples were separated by SDS-PAGE and entire gel lanes, including empty vector controls, were subjected to an in-gel digestion step. The resulting peptides were then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a LTQ linear ion trap mass spectrometer. After database searching, an in-house software application was used to identify proteins that were unique to the Ubxd1- containing samples. A number of these proteins were selected for validation experiments using standard co-immunoprecipitation and immunofluorescence techniques. Results Using a targeted interaction mapping approach, numerous potential interaction partners of Ubxd1 were identified, including components of an inhibitory complex called the mitotic checkpoint complex. Although the Flag- and HA- interaction datasets displayed minimal overlap among the majority of proteins identified, components of the mitotic checkpoint complex were consistently observed. Co-immunoprecipitation experiments confirmed that these proteins were specifically purified only in the presence of Ubxd1. Co- localization experiments also confirmed that these proteins were present in the same physical location within the cell as Ubxd1. Finally, results from several Ubxd1 knock-down experiments demonstrated an increase in polyploidy, which is a phenomenon known to be associated with perturbation of the mitotic checkpoint complex. Quantification of Protein Complex Components in Interaction Networks 1,2 1,2 , 1,2,3,4 1,2 Alexander Wepf , Timo Glatter , Ruedi Aebersold and Mat thias Gstaiger 1 Institute of Molecular Syste ms Biology, ETH Zurich, Zurich, Switzerland. 2Competence Center for Systems 3 Physiology and Metabolic Diseases, ETH Zurich, 8093 Zurich, Switzerland. Faculty of Science, University of Zurich, Zurich, Switzerland, 4Institute for Systems Biology, Seattle, WA, USA Introducti on Protein complexes represent major functional units Innovative aspects for the control of biological processes. • absolute quantification of protein complex Understanding on how proteins are organized into components in large scale AP -MS studies protein complexes at a global scale is becoming • no need for a large set of heavy peptides increasingly important for a systems level conception of cellular processes. Until today, protein interaction networks are a purely qualitative representation of com plex interactions, eve n though affinity -purification coupled to mass spectrometry (AP -MS), the method of choice for network analysis, could in principle be applied in a quantitative fashion. Methods Here a method is presented w hich combines the advantages of isotope -labelled pept idesfor absolute quantificationwith lab le-free quantitation. First, a single heavy peptide of an engineered amino acid sequence in the affinity tag is used for absolutequantification of the bait protein . Second, relative quantification on the intensities of the M S1 signal is performed: Thereby, t he peptides of the bait protein serve as sta ndard peptides to quantify the absolute amoun ts of the same protein in the other affinity -purificat ions. Results Particularly highly connected network s like the human protein phosphatase 2A network are suitable for this combined quantification method. In a large number of interactions the absolute amount of bait as well as the absolute amount of prey present after an affinity purification can be quantified concurrently without neither the need for knowing or predicting proteotypic peptides nor the need of an expensive se t of heavy peptides. Quantitative network information generated in such a manner allows network model ling and real complex prediction with much higher accuracy. Protein associations can be classified into abundant and stable associations versus interactions present in sub -stoichiometric amounts or of transient stability. Furthermore complex composition changes can be monitored and changes in seco ndary modifications can be normalized to the pro tein abundance. Identification of putative complex I assembly factors by Blue Native Liquid Chromatography MS/MS J.C.T. Wessels, R.O. Vogel, L.P. van den Heuvel, R.J. Rodenburg, L.G. Nijtmans, J.A.M.Smeitink, M.H. Farhoud Nijmegen Center for Mitochondrial Disorders, Laboratory of Pediatrics and Neurology, Radboud University Medical Center Nijmegen, Geert Grooteplein 10, 6500HB Nijmegen,The Netherlands Introduction. Analysis of protein (sub)complexes proteins identified by this approach is C6ORF66 by Blue Native (BN) electrophoresis produces which was recently published as an assembly invaluable information about subunit composition factor for complex I (1). This protein showed clear of complexes and assembly intermediates. The comigration with CI assembly intermediates on analysis of co-migrating proteins is often both gradients (figure 1) which was validated by performed by antibody detection or mass westernblot results using NDUFS3 and C6ORF66 spectrometric (MS) identification in a single or two antibodies. dimensional separation. Unfortunately, these approaches are limited by the availability of Innovative aspects. antibodies or separation capability for MS analysis. • First application of LC MS protein Given these limitations we hypothesized to use correlation profiling to blue native Liquid Chromatography (LC) MS/MS as a second electrophoresis. dimension separation, identification and quantitation method. • Most extensive analysis of CI assembly intermediates to date adding new insights Methods. To analyze the mitochondrial into their exact composition. complexes we separated proteins on Blue Native gels using 4-12% and 5-15% acrylamide gradients. • Identification of multiple putative CI Each gel lane was cut into 24 slices and analyzed assembly factors in duplo by LC MS/MS. Protein identifications were performed by database searching. Quantitative References: data were extracted as EMPAI, extracted ion 1. Saada et al, C6ORF66 is an assembly chromatograms (XIC), and by accurate mass & factor of mitochondrial complex I; Am. J. retention time (AMT) approach to evaluate their Hum. Genet. 82, 32-38 applicability to analyze protein migration patterns. Identification of putative complex I assembly factors was achieved by searching the dataset for proteins that comigrate with complex I assembly intermediates via template matching (Pearson correlation values). Migration patterns were validated by 2D BN SDS-PAGE immunodetection. Results. Evaluation of quantitation methods showed that all approaches appear applicable to study migration patterns on BN gels. Based on close evaluation of the data we decided to primarily use XIC values and only perform AMT for a relatively low number of proteins of interest. Analysis of complexes from the oxidative phosphorylation system showed focusing of respective subunits at the expected migration distances. Close evaluation of complex I (CI) subunits revealed comigration of NDUFS subunits 2,3,7 and 8 in well separated subcomplexes of smaller size than holocomplex I. Alignment of the migration patterns of NDUFS2 and 3 with westernblot results identified these subcomplexes as known assembly intermediates of CI. To find novel CI assembly factors we used the CI assembly intermediates pattern to screen the Figure 1. Normalized migration patterns as determined datasets for comigrating proteins. Using an by LC MS of C6ORF66 (yellow) and average template empirically determined Pearson correlation score pattern (blue) for 4-12% and 5-15% acrylamide blue of 0.7 we were able to specifically look for native gels. Assembly intermediates are respectively in candidate assembly factors. One of the candidate slices 8-24 and 6-24 for the 4-12% and 5-15% gradients. Contrasting Proteome Biology and Functional Heterogeneity of the 20S Proteasome Complexes. GW. Young. AV. Gomes, Y. Wang, P. Ping. Department of Medicine and Physiology, University of California, Los Angeles. Los Angeles, CA. 90024 United States of America Introduction: The 20S proteasome complexes are Results: Our results revealed an organ specific the major contributors to the intracellular protein molecular organization of the 20S proteasomes degradation machinery in mammalian systems. with distinguished patterns of post-translational Systematic administration of proteasome inhibitors modifications as well as unique complex assembly utilized to combat diseases (e.g. cancer) has characteristics and associating proteins. shown targeted actions as well as adversary Furthermore, the proteome diversities are effects. The latter was attributed to a lack of concomitant with a functional heterogeneity of the understanding pertaining to organ specific proteolytic patterns exhibited by these two organs. responses to these inhibitors and the potential In particular, the liver and heart displayed distinct diversity of proteome biology of these complexes activity profiles to two proteasome inhibitors, in different tissues. epoxomicin and Z-Pro-Nle-Asp-H. Finally, heart and liver demonstrated contrasting regulatory Methods: Accordingly, we conducted a proteomic mechanisms to the associating partners of these study to characterize the 20S proteasome proteasomes in respect to protein phosphatase 1 complexes and their postulated organ-specific (PP1) and casein kinase II (CKII). CKII decreased responses in liver and heart. The cardiac and all three proteolytic activities of the hepatic 20S hepatic 20S proteasomes were isolated from the proteasomes and had no detectable effect in the same mice with identical genetic information. heart, while PP1 enhanced just the β5 cardiac 20S Purified proteasome samples were resolved by activity, with no detectable effect in the liver. Taken either denaturing or native electrophoresis. Bands together, these findings illustrate differential were tryptically digested and analyzed on an LTQ proteome biology in an organ specific fashion, i.e. mass spectrometer. We then examined the the unique assembly and functional heterogeneity molecular composition, complex assembly, post- of the mammalian 20S proteasome complexes. translational modifications and associating partners of these proteasome complexes. Proteolytic assays of the 20S proteasomes investigated all three proteolytic activities as they differed between the heart and liver and in response to manipulation of the suspect associating partners.
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