exosomes are extracellular vesicles 30100 
 nm in diameter 
 that are shed by most cells . they were first observed 
 in 1987 in maturing reticulocytes , and 
 recently they have gained attention as agents of intercellular communication and as potential prognostic tools . in previous studies , we interrogated the content 
 of myeloid - derived suppressor cells ( mdsc ) and exosomes shed by mdsc . we focused on these cells because of their widespread presence in 
 most cancer patients and their critical role in promoting tumor progression 
 through their inhibition of innate and adaptive antitumor immunity . we have reported that exosomes shed by mdsc contain 
 proinflammatory molecules that drive the accumulation and immune suppressive 
 potency of mdsc and macrophages , respectively , and have identified 
 the specific proteins responsible for these bioactivities . on the basis of these and other potential functions , 
 there is global interest in the nature of the protein cargo carried 
 by exosomes . exosomes are formed by a series of intracellular events initiated 
 by the invagination of the plasma membrane to form endosomes . within endosomes , these luminal vesicles and their cargo are then incorporated into 
 the lysosome for degradation , recycled to the plasma membrane , or 
 exocytosed as exosomes . ubiquitination has been shown to signal both 
 the internalization of surface proteins and the sorting of endosomal 
 proteins into luminal vesicles . given the strategic role of ubiquitination in intracellular 
 protein 
 trafficking , the present study seeks to confirm the presence and identify 
 ubiquitinated proteins in exosomes derived from mdsc . a previous study 
 using western blot analyses indicated that exosomes and their parental 
 cells contain distinct populations of ubiquitinated proteins ; however , the conjugated proteins were not identified . identification of the conjugated proteins allows assignment of their 
 original locations in the parent cell , their original cell functions , 
 and the range of protein sizes and pi values and may contribute to 
 understanding the complexity of the endosomal pathway . in addition , 
 enriching for ubiquitinated proteins allows detection of proteins 
 that may be relatively low in abundance but play an important role 
 in exosome structure and function and that contribute to mdsc function . in the present work , ubiquitinated proteins have been recognized 
 in mdsc - derived exosomes by identifying peptides that carry glycinylglycine - modified 
 lysine residues as remnants of the ubiquitin carboxyl terminus . additionally , 
 we have used gene ontology annotations and the uniprot database to 
 look for trends in the source , function , size , and pi values of the 
 ubiquitinated species . mass spectrometry - based bottom - up proteomics 
 has proven to be a 
 powerful tool for recognizing ubiquitinated tryptic peptides and identifying 
 sites of ubiquitination on these peptides and their related proteins . tryptic digestion of ubiquitinated proteins cleaves ubiquitin at r74 , 
 which leaves two glycine residues on the modified lysine of the substrate 
 peptide . enrichment of ubiquitinated 
 proteins from mdsc - derived exosomes was accomplished using two immunoprecipitation 
 steps : immunoprecipitation of ubiquitinated proteins followed by tryptic 
 digestion and immunoprecipitation of peptides containing glycinylglycine - modified 
 lysine residues . in a parallel workflow , immunoprecipitation of ubiquitinated 
 proteins was followed by 1d gel electrophoresis and in - gel digestion . 
 in each case , ms / ms 
 and a bioinformatic search program that allowed for the variable modification 
 of glycinylglycine - modified lysine residues ( kgg ) . here these ubiquitinated proteins constitute 
 a small subset of proteins in these exosomes , originate from a diversity 
 of subcellular locations , and have a variety of functions . balb / c mice were injected 
 in the mammary fat pad with approximately 7000 wild - type 4t1 mammary 
 carcinoma cells stably transfected to express interleukin-1 
 ( il-1 ) . when tumors were greater than approximately 8 mm in 
 diameter ( about 34 weeks after initial inoculation ) , mdsc 
 were harvested from the blood , stained with fluorescently labeled 
 monoclonal antibodies against markers of mdsc ( gr1 and cd11b ) , and 
 analyzed by flow cytometry . cell populations 
 that were greater than 90% gr1cd11b were used 
 in all experiments . for each experiment , a total of about 1  10 mdsc were pooled from 2 to 
 3 mice . the umbc and umcp institutional animal care and use committees 
 approved all procedures with animals and animal - derived materials . mdsc were plated in serum - free hl-1 medium 
 ( biowhittaker , walkersville , md ) and maintained at 37 c with 
 5% co2 . after 18 h , the cultures were centrifuged at 805 g for 5 min ( eppendorf 5810 rotor , eppendorf , hamburg ) , 
 the pellets were discarded , and the supernatants were centrifuged 
 at 2090 g for 30 min ( sorvall rc5c , ss34 rotor , dupont , 
 wilmington , de ) . the supernatants were then ultracentrifuged at 100  000 g for 20 h at 10 c ( beckman l8 , sw40ti rotor , beckman , 
 pasadena , ca ) . the supernatants were discarded , and the pellets containing 
 the exosomes were resuspended in pbs . exosomes were lysed in an optimized lysis buffer of 8 m urea in 
 50 mm ammonium bicarbonate with 50 m of deubiquitinase inhibitor 
 pr-619 ( lifesensors , malvern , pa ) and 1% of a protease inhibitor cocktail 
 ( sigma - aldrich , st . they were centrifuged at 14  000 g for 30 min with a 3 kda molecular weight cut off filter , 
 and the supernatants were discarded . after lysis , the buffer was diluted to 0.8 m urea in 50 mm ammonium 
 bicarbonate . protein content before and after immunoprecipitation 
 was measured by the quick start bradford assay ( bio - rad , hercules , 
 ca ) . ubiquitinated 
 proteins were enriched using protein a - sepharose 4b beads ( invitrogen , 
 carlsbad , ca ) that had been incubated with anti - ubiquitin antibody 
 3933 ( cell signaling technology , danvers , ma ) in a 1:600 dilution 
 with rotation for 4 h at 4 c . excess antibody was removed from 
 the beads by washing with 0.8 m urea in 50 mm ammonium bicarbonate 
 and centrifuging three times at 3000 g for 2 min . one - hundred micrograms of exosome lysate was added to the sepharose 
 bead slurry and incubated with rotation overnight at 4 c . the sepharose bead slurry was washed with 50 mm ammonium 
 bicarbonate and centrifuged at 1000 g for 5 min to 
 remove nonspecifically bound proteins . bound proteins were eluted 
 by incubating the sepharose bead slurry in 0.2 m glycine , ph 2.6 , 
 for 1 h at 4 c and collected via centrifugation at 13  000 g for 5 min . enriched fractions 
 of ubiquitinated exosomal proteins were subsequently processed either 
 by tryptic digestion in gel or in solution and immunoprecipitation 
 of peptides with glycinylglycine - modified lysine residues . proteomic studies 
 were conducted on exosomal proteins enriched for 
 ubiquitin conjugates by immunoprecipitation . three biological replicates 
 were resuspended in 2% sds , 5% -mercaptoethanal , and 62.5 mm 
 tris hcl and reduced at 90 c for 5 min . the samples were then 
 loaded onto 816% polyacrylamide gels ( bio - rad ) and subjected 
 to electrophoresis for approximately 50 min at 200 v , 15 ma , and 50 
 w. the gels were stained using coomassie blue ( 40% methanol , 20% acetic 
 acid , 0.1% m / v coomassie blue reagent 250 ; thermo scientific , san 
 jose ca ) stain and then cut into 13 slices . after destaining , tryptic 
 digestion was performed on each gel slice overnight at 37 c . the extracted tryptic peptides were resuspended 
 in 0.1% formic acid for injection into the lc  ms / ms . enriched fractions of ubiquitinated exosomal proteins 
 from five biological replicates were frozen , lyophilized , and resuspended 
 in 50 mm ammonium bicarbonate . proteins were reduced with 20 mm dithiothreitol 
 for 30 min at 56 c and alkylated with 10 mm methylmethanethiosulfonate 
 for 45 min . one microgram of trypsin was added to each fraction , and 
 digestion was performed overnight at 37 c . as a positive control , 
 a ubiquitin dimer linked with an isopeptide bond at k48 ( life sensors , 
 malvern , pa ) peptides with glycinylglycine - modified lysine residues were enriched 
 using protein a - sepharose 4b beads coupled to anti - diglycyl - lysine 
 antibody gx41 ( millipore , billerica , ma ) using the same procedure 
 as that with the anti - ubiquitin antibody , except the anti - diglycyl - lysine 
 antibody was prepared at a 1:1000 dilution . the fractions of immunoprecipitated 
 ubiquitinated proteins were added to the sepharose bead slurry and 
 incubated with rotation overnight at 4 c . the unbound fraction 
 was removed via centrifugation at 500 g for 5 min . the sepharose bead slurry was washed with 50 mm ammonium bicarbonate 
 and centrifuged at 1000 g for 10 s to remove nonspecifically 
 bound peptides . bound peptides were eluted by incubating the sepharose 
 bead slurry in 0.2 m glycine , ph 2.6 , for 1 h at 4 c and collected 
 via centrifugation at 13  000 g for 5 min . the 
 elution was repeated , and the two elution fractions were combined . 
 prior to lc ms / ms analysis , all fractions were desalted with 
 c18 toptip spin columns ( glygen , columbia , md ) and resuspended in 
 100 l of 0.1% formic acid . all fractions were subjected to one - dimensional 
 gel electrophoresis on an 816% criterion precast gel ( bio - rad ) 
 at 200 v , 50 ma , and 15 w for 56 min , followed by transfer to a pvdf 
 membrane ( emd millipore , billerica , ma ) at 100 v , 350 ma , and 35w 
 for 1 h. free ubiquitin , polyubiquitin , and ubiquitinated proteins 
 were detected by blotting with anti - ubiquitin antibody 3933 ( cell 
 signaling technology ) followed by anti - mouse igg - hrp ( cell signaling 
 technology ) . protein bands were visualized with an image lab system 
 ( bio - rad , hercules , ca ) using the gel - doc program ( kodak molecular 
 imaging systems ) and the supersignal west dura chemiluminescent substrate 
 ( thermo fisher scientific , waltham , ma ) . exosomal histones were extracted 
 using the epiquick total histone extraction kit ( epigentek , farmingdale , 
 ny ) according to the manufacturer s instructions and analyzed 
 for ubiquitination via western blotting with anti - ubiquitin antibody 
 3933 as previously described . ms / ms analyses were performed 
 on a shimadzu prominence 
 nano hplc ( shimadzu scientific instruments , columbia , md ) in - line 
 with an ltq orbitrap xl ( thermo fisher scientific ) . a 10 l 
 aliquot of tryptic peptides was injected onto an acclaim pepmap 300 
 c18 precolumn ( dionex , sunnyvale , ca ) followed by desalting with 10% 
 solvent a ( 97.5% h2o , 2.5% can , and 0.1% formic acid ) for 
 20 min . peptides were fractionated on a c18 analytical column ( 150 
  0.15 mm , 300  , grace davidson discovery sciences , deerfield , 
 il ) with a linear gradient increasing from 0 to 40% solvent b ( 97.5% 
 acn , 2.5% h2o , and 0.1% formic acid ) in 85 min , followed 
 by an increase from 40 to 85% solvent b in 20 min . precursor scans were acquired in the orbitrap with a resolution 
 of 30  000 at m / z 400 . in 
 each cycle , the nine most abundant ions were selected for fragmentation 
 by collisional induced dissociation , and product ion scans were acquired 
 in the ltq . a dynamic exclusion of 1 repeat count over 180 s was used . peptide and protein identifications were made by the peparml meta - search engine against the uniprot mouse database ( july 2014 ) . for the in - gel digestion , all peptide identifications were filtered 
 at 10% spectral fdr , and proteins were required to be supported by 
 at least 2 unshared peptides , bounding a protein fdr at 1% . for the 
 two - step immunoaffinity enrichment of glycinylglycine - modified lysine - containing 
 peptides , all peptide identifications were filtered at a 5% spectral 
 fdr . fixed modifications listed methylthio modification of cysteine , 
 and proteins with at least one peptide with a 
 lysine residue tagged with glycinylglycine were considered to be ubiquitinated . 
 when a protein identification was based on a single peptide from the 
 double immunoaffinity workflow , subcellular location 
 and function assignments of the identified proteins were made using 
 the protein information resource go slim ( http://pir.georgetown.edu ) using uniprot gene ontology annotations ( july 2014 ) . western blotting was used 
 to determine if mdsc - derived exosomes contain ubiquitinated proteins . a general ubiquitin antibody was used as well as antibodies that recognize 
 k48 and k63 linkages in ubiquitin chains . figure 1 confirms the presence of ubiquitinated proteins , including 
 proteins with k48- and k63-linked branched ubiquitins . consequently , 
 mass spectrometry - based proteomic strategies were used to identify 
 conjugated proteins . in - gel digestion of immunoprecipitated ubiquitin - conjugated 
 proteins table 1 lists 16 ubiquitinated proteins identified from glycinylglycine - tagged 
 peptides recovered from tryptic digestion in gel . table 2 lists 38 proteins identified from glycinylglycine - modified 
 peptides isolated after tryptic digestion in solution . the experimental 
 design , with immunoaffinity isolation of glycinylglycine - modified 
 peptides , leads to several protein identifications that are each based 
 on a single tagged peptide and thus are less reliable than those listed 
 in table 1 . these are listed in table 2 , and annotated spectra are provided in supporting information . antibodies 
 used are ( left ) anti - ubiquitin 3933 , ( middle ) anti - k-48-linked polyubiquitin , 
 and ( right ) anti - k-63-linked polyubiquitin . indicates that the protein was also 
 identified in the in - gel digestion . to report a protein as being ubiquitinated , reliable 
 identification 
 was required of at least one peptide containing a kgg residue . initial identifications were made by the peparml meta - search engine 
 ( see experimental section ) , and all candidate 
 tandem mass spectra were confirmed manually ( supporting 
 information figure s1 ) . a combined total of 65 tryptic peptides 
 containing modified lysines correspond to 50 ubiquitinated proteins 
 ( supporting information table s1 ) . only 
 10 of the 50 proteins identified were previously reported in an mdsc - derived 
 exosome lysate , demonstrating that enrichment 
 for ubiquitinated proteins enabled the identification of low - abundance 
 proteins in exosomes . as suggested by the western blots in figure 1 , several histones were also observed to be conjugated 
 at multiple unique and nonoverlapping sites , and the pro - inflammatory 
 high mobility group protein b1 ( hmg b1 ) was found to be ubiquitinated . the glycinylglycine - lysine sites identified experimentally were compared 
 to ubiquitination sites predicted in silico by a ubiquitination prediction 
 tool , ubiprober . of the 65 peptides identified , 
 the glycinylglycine - lysine sites in 42 peptides are ubiquitination 
 sites predicted with probabilities > 0.7 , ubiprober s confidence 
 level . tables 1 and 2 also 
 present the glycinylglycine - tagged peptides , where it can be seen 
 that 15 of the 65 peptides have been formed by tryptic cleavage at 
 gg - derivatized lysine residues . this unexpected tryptic cleavage has 
 been observed previously by others , and a control 
 experiment was also carried out to confirm its occurrence under the 
 conditions of the present investigation . a commercial ubiquitin dimer 
 linked with an isopeptide bond at k48 was subjected to tryptic digestion 
 as described in the experimental section . both the expected peptide lifagkggqledgr and the irregular 
 peptide lifagkgg were identified by tandem mass spectrometry 
 in approximately a 3:1 ratio ( data not shown ) . several comparisons were made between the ubiquitinated 
 proteins 
 and a larger set of 412 proteins identified in an earlier study of 
 mdsc exosome lysates . figure 2 presents a comparison of the uniprot - derived locations 
 of exosome proteins referenced to parental mdsc and illustrates an 
 increased percentage of nuclear proteins in the ubiquitinated cohort 
 and significantly lower percentages of cytosolic and plasma membrane 
 proteins . the ubiquitinated nuclear proteins include nine histones 
 and isoforms as well as other nucleic acid binding proteins ( transcription 
 factor a , mitochondrial , density regulated protein , transcription 
 initiation factor tfiid subunit 3 , and protein bodl1 ) ( tables 1 and 2 ) . it should be noted 
 that histones , especially linker histones such as the histone h1 family , 
 have been reported to be located in the cytoplasm and cell surface 
 as well as the nucleus and that several 
 histones are already known to be ubiquitinated , e.g. , refs ( 3032 ) . the observation of ubiquitinated histones was confirmed 
 by western blotting using anti - ubiquitin antibody 3933 on the histone 
 fraction recovered using a total histone extraction kit ( see experimental section ) ( data not shown ) . figure 2 also indicates that 12 of the ubiquitinated proteins 
 have no assigned cellular location . protein locations assigned to mdsc - derived 
 exosomal lysate ( 412 
 proteins ) in gray and the ubiquitinated cohort ( 50 proteins ) in black . gene ontology annotations and the uniprot database were used 
 to 
 compare distributions of protein sizes ( without ubiquitin ) , grand 
 average of hydropathicity ( gravy ) scores , and isoelectric points ( pi ) 
 of the 50 ubiquitinated proteins identified . the intact masses of 
 the proteins are somewhat evenly distributed between 11 and 327 kda 
 ( excluding ubiquitination ) . the gravy scores and pi distributions 
 are shown in figure 3 , panels a and b , respectively . although both sets of data illustrate a wide range of gravy scores 
 ( 0.081 to 1.627 ) and pi values ( 4.72 to 11.71 ) , proteins 
 that are ubiquitinated in mdsc - derived exosomes tend to cluster as 
 hydrophilic and basic proteins . seventy - two percent of the proteins 
 identified have a gravy score less than 0.5 , and 50% of the 
 proteins have a pi greater than 9.00 . ( ubiquitin is not included in 
 these calculations ; its monomer has a gravy score of 0.489 
 and a pi value of 6.56 . ) the bias toward a high ph is consistent with 
 observations by chen and co - workers , who 
 report a greater abundance of positively charged amino acids in ubiquitinated 
 proteins . distribution of ( top ) grand average of hydropathicity score ( gravy ) 
 and ( bottom ) isoelectric point of mdsc - derived exosomal lysate ( 412 
 proteins ) in gray and the ubiquitinated cohort ( 50 proteins ) in black . among the combined cohort of 50 
 proteins , 34 have not been previously 
 reported to be ubiquitinated ( tables 1 and 2 ) . among these , sorting nexin 13 has been observed 
 to participate in endosomal trafficking of ubiquitinated proteins . identification of two ubiquitinated keratins 
 is consistent with the proposed role of protein aggregation in invagination , 
 the initial step of exosome formation . other ubiquitinated 
 proteins that are thought to play important roles in endosome and 
 exosome formation include leucine zipper ef hand - containing transmembrane 
 protein 1 ( letm1 ) and endoplasmin . although the functions of the ubiquitinated 
 proteins are not known , these two unconjugated proteins participate 
 in transporting and maintaining the high luminal concentration of 
 ca required for optimal exocytosis of exosomes . on the basis of protein 
 assay results , approximately 10% of the 
 mdsc - derived exosome lysate comprises ubiquitinated proteins . tandem 
 mass spectrometry coupled with immunoprecipitation has been used successfully 
 to isolate and identify 50 ubiquitinated proteins from mdsc - derived 
 exosomes and to determine their positions of conjugation . five of 
 these are associated with formation of endosomes and exosomes , consistent 
 with earlier proposals . the skew of pi values toward basicity in the 
 conjugated cohort of exosomal proteins may contribute to their concentration 
 and retention by the progressive acidification that has been documented along the endosomal pathway . the presence of 
 ubiquitinated histones in these exosomes should be considered in the 
 context of heightened interest in extracellular histones and their 
 proinflammatory activity . hmg b1 is another pro - inflammatory 
 mediator of particular interest since it was recently established 
 as a driver of mdsc accumulation and suppressive potency . interestingly , s100 a8 and s100 a9 , exosomal 
 pro - inflammatory proteins previously demonstrated to contribute to the bioactivity of mdsc , have not been 
 identified with a glycinylglycine - lysine modification , even though 
 they are quite abundant and readily identified in the exosome data 
 sets .
we 
 provide evidence at the molecular level that ubiquitinated proteins 
 are present in exosomes shed by myeloid - derived suppressor cells ( mdsc ) . 
 ubiquitin was selected as a post - translational modification of interest 
 because it is known to play a determinant role in the endosomal trafficking 
 that culminates in exosome release . 
 enrichment was achieved by two 
 immunoprecipitations , first at the protein level and subsequently 
 at the peptide level . 
 fifty ubiquitinated proteins were identified 
 by tandem mass spectrometry filtering at a 5% spectral false discovery 
 rate and using the conservative requirement that glycinylglycine - modified 
 lysine residues were observed in tryptic peptides . 
 thirty five of 
 these proteins have not previously been reported to be ubiquitinated . 
 the ubiquitinated cohort spans a range of protein sizes and favors 
 basic pi values and hydrophobicity . 
 five proteins associated with 
 endosomal trafficking were identified as ubiquitinated , along with 
 pro - inflammatory high mobility group protein b1 and proinflammatory 
 histones .