posttranslational protein modifications 
 ( ptm ) , such as phosphorylation , 
 are difficult to quantify because they are highly dynamic , present 
 on proteins at low levels , and often of low stoichiometries . quantitative 
 analysis of ptm has been achieved using liquid chromatography tandem 
 mass spectrometry ( lc  ms / ms ) . global quantification of phosphorylation 
 or other ptm typically employs isotope labeling by chemical derivatization 
 ( e.g. , itraq ) or metabolic incorporation of stable isotope - labeled 
 amino acids in cell culture ( silac ) . stable isotope dilution provides the highest 
 precision for quantitative ms studies ; however , it requires 
 both synthesis and amino acid analysis for each peptide of interest . the cost associated with sid quanatitive analyses becomes cost - prohibitive 
 when many targets need to be analyzed . an alternative to stable 
 isotope labeling strategies is the use 
 of commonly performed and accepted  label free these methods fall broadly into two groups , spectral counting 
 and integrating ms peak areas . although spectral counting 
 methods compares favorably to stable isotope labeling in both precision 
 and accuracy for quantifying proteins in shotgun proteomics analyses , the sampling of individual modified peptide spectra by  data - dependent 
 ms / ms  is insufficient in capturing enough spectra to use this 
 method for quantification at the peptide level . quantification by 
 integrated ms signals for specific peptide ions is performed 
 by extraction of selected ion chromatograms from lc  ms data 
 sets . although a higher resolution instrument , such as 
 an ltq - orbitrap or q - tof , may improve the background for an ms signal , this instrumentation may not be readily available 
 for these experiments . in addition , the ms signal does 
 not distinguish between sites of differentially modified peptides , 
 particularly problematic when the peptides can not be chromatographically 
 resolved , and ms signals for lower level peptides ( such 
 as modified peptiedes ) are frequently hindered because of significant 
 background noise . ion traps also have the ability to perform ms , which is not possible with q - tof intruments . in many cases , analysis of ptm is done on purified proteins or simple mixtures , such 
 as immunoprecipitates , expressed proteins , or proteins purified by 
 chromatography or electrophoresis . in such cases , the need to quantify 
 a stoichiometric change in a modification frequently follows the initial 
 identification of modified forms . since the modified form has been 
 identified , the ms / ms spectral characteristics of the modified peptides 
 of interest are known . in this context , the goal is targeted quantitation 
 of changes to specific proteins , rather than a quantitative global 
 survey . here we describe the validation of a label - free approach to 
 measure quantitative changes in modifications to specific proteins . ms / ms analysis with extracted 
 selected reaction monitoring ( pseudo - srm or psrm ) using a linear ion - trap 
 mass spectrometer to enable selective detection and further quantification 
 of modified peptides . we have implemented full support for this workflow 
 in version 1.1 of the software tool skyline ( http://proteome.gs.washington.edu/software/skyline/ ) , a widely used , freely available tool for srm anlaysis . to correct for run - to - run variations in signal 
 intensities , psrm signals for the target peptides are normalized to 
 signals from unmodified reference peptides from the same protein , 
 which we termed the internal reference peptide ( irp ) method . although 
 we and others have described similar quantification methods previously , here we performed proof - of - principle experiments that allowed us 
 to validate the method , define its performance characteristics and 
 compare them to stable isotope dilution , which is the accepted we further assessed the 
 performance of psrm using both ms / ms and ms data for quantification . we describe the application of the irp method to analyze phosphorylated 
 forms of epidermal growth factor receptor ( egfr ) , an extensively characterized 
 receptor tyrosine kinase and a target for several clinically used 
 anticancer drugs . our data demonstrate the proficiency of the irp 
 method to quantify site - specific changes in egfr phosphorylation in 
 response to modulation by egf and the two tyrosine kinase inhibitors , 
 cetuximab and gefitinib . acetonitrile and hplc - grade 
 water were from mallinckrodt baker ( phillipsburg , nj ) , and 98% pure 
 formic acid was from emd ( darmstadt , germany ) . trypsin gold was purchased 
 from promega ( madison , wi ) , dithiothreitol ( dtt ) from pierce ( rockford , 
 il ) , and iodacetamide was from sigma ( st . the a431 cell 
 line was obtained from atcc ( manassas , va ) , improved mem media , and 
 pbs were purchased from invitrogen - gibco ( auckland , nz ) . media supplement , 
 fetal bovine serum , was from atlas biologicals ( fort collins , co ) . 
 for western blotting , primary antibodies forphosphotyrosine site 1172 , 
 phosphotyrosine site 998 and egfr were purchased from cell signaling 
 technology ( danvers , ma ) . primary antibodies were detected using antirabbit 
 and antimouse secondary antibodies conjugated with fluorophore 680 
 from invitrogen ( carlsbad , ca ) and imaged using the li - cor odyssey 
 imager system with 3.0 application software ( lincoln , ne ) . all gels 
 ( nupage ) , western blot membranes and gel reagents ( lds , pvdf membrane , 
 and simplyblue safestain ) were purchased from invitrogen ( carlsbad , 
 ca ) . individual components of the netn lysis buffer , protease inhibitor 
 cocktail and phosphatase inhibitor cocktail ( see below ) were purchased 
 from sigma ( st . beads for immunoprecipitations , protein 
 a and protein g were purchased from thermoscientific ( rockford , il ) 
 and roche ( indianapolis , in ) , respectively . synthetic phosphorylated 
 peptides , drvpyihpf and iknlqpsldpsh , 
 were purchased as part of the phosphopeptide standard i from protea 
 biosciences ( morgantown , wv ) . bovine serum albumin ( bsa ) was purchased 
 from thermoscientific ( rockford , il ) . n2-lysine 
 from egfr ( y998mhlpsptdsnfpyr , y1110rpagsvqnpvpyhnqplnpapsr , y1172gshqisldnpdpyqqdffpk , and y1197gstaenaepylr ) were purchased from new england peptide , llc ( gardner , 
 ma ) at 95% chemical purity based on amino acid analysis . carlos arteaga , both from vanderbilt  ingram 
 cancer center ( nashville , tn ) . synthetic phosphopeptide 
 mixture ( drvpyihpf and iknlqpsldpsh ) 
 was resuspended in 0.1% formic acid to a concentration of 500 pmol 
 ml , and peptides were spiked into 100 l 
 of 6.0 g ml ( 6 ng of bsa ) tryptic digest 
 of bsa at concentrations ranging from 0.01 to 2.0 fmol ng of bsa ( which corresponds to 0.06412.8 fmol l ) in 0.1% formic acid . the bsa tryptic digest was 
 performed on 200 g of 2 mg ml of bsa . the 
 sample was diluted in ammonium bicarbonate , reduced in 45 mm dtt at 
 55 c for 20 min , alkylated in the dark at room temperature for 
 20 min using 100 mm iodoacetamide , and digested with 4 g of 
 trypsin overnight at 37 c . an aliquot of this digest was diluted 
 with 0.1% formic acid to a final concentration of 6.0 g ml . a human epithelial carcinoma cell line 
 ( a431 ) was cultured in 150 mm culture dishes in improved mem supplemented 
 with 10% fetal bovine serum at 37 c in 5% co2 . a431 
 cells were grown to 6070% confluency prior to harvesting 
 ( control or treatment ) . all treated cells were serum - starved ( 18 h ) , 
 followed by treatment with 30 nmol epidermal growth factor ( egf ) for 
 20 min or incubated with either 10 g ml cetuximab or 500 nmol gefitinib for 30 min followed by subsequent 
 stimulation with egf for 20 min . cells were harvested on ice with 
 mg and cl - free pbs supplemented with a phosphatase inhibitor cocktail 
 ( 1 mm sodium fluoride , 10 mm -glycerophosphate , 1 mm sodium 
 molybdate , and 1 mm activated sodium orthovanadate ) , pelleted by centrifugation 
 at 250 g , flash - frozen , and stored at 80 
 c . cell pellets were lysed by resuspension in netn lysis buffer ( 50 
 mm tris - hcl , ph 7.5 , 150 mm nacl , 1% igepal , and 5% glycerol ) containing 
 protease inhibitors ( 0.5 mm 4-(2-aminoethyl)benzenesulfonyl fluoride 
 hydrochloride , 10 mm aprotinin , 1 mm leupeptin , 1.5 m e-64 , 
 5 m betastatin , and 1 m pepstatin a ) and the phosphatase 
 inhibitor cocktail and incubated on ice for 25 min prior to mechanical 
 lysis by sonication . after cell lysis , suspensions were cleared by 
 centrifugation at 9400 g for 5 min . the bicinchoninic 
 acid assay ( protein standard was bovine serum albumin ) a 100 g 
 aliquot of total cell lysate was collected as input control and combined 
 with 4x lds buffer and dtt for a final concentration of 1x and 50 
 mm , respectively . a 3 mg portion of the remaining cleared lysate was 
 incubated at 4 c for 1.5 h with cetuximab at a ratio of 5 g 
 of cetuximab for every 1 mg of cell lysate . a 30 l portion 
 of pre - equilibrated protein a and protein g resin ( 1:1 v / v ) was added 
 to the suspension and incubated with the lysate for 45 min at 4 c . the supernatant was removed and the resin 
 washed three times with netn lysis buffer . protein(s ) were eluted 
 by treating beads for 5 min at 85 c in 2x lds buffer and 50 
 mm dtt . gels were either prepared for western blot 
 by transferring proteins to pvdf membrane or stained ( for 1 h ) using 
 simplyblue safestain followed by destaining in deionized water overnight . targeted ms analysis was performed on the digested egfr gel regions . briefly , the egfr protein bands were excised and rinsed with 100 mm 
 ammonium bicarbonate ( ph 8.0 ) . gel pieces were reduced with 50 mm 
 dtt at 60 c for 30 min , followed by alkylation with 100 mm iodoacetamide 
 in the dark at ambient temperature for 20 min , and digested with 200 
 ng trypsin overnight at 37 c . peptides were extracted from the 
 gel three times with 60% acetonitrile/0.1% formic acid ( v / v ) . peptides 
 were concentrated in vacuo ( speedvac concentrator , thermo - fisher , 
 waltham , wa ) and reconstituted in 30 l of 5% acetonitrile and 
 0.1% formic acid with 12.5 fmol l of isotopically 
 labeled peptides spiked in for lc  psrm  ms analysis . lc  psrm  ms 
 and ms analyses were performed on a thermofisher 
 ltq velos ( san jose , ca ) mass spectrometer equipped with an eksigent 
 nano-1d plus hplc and as-1 autosampler ( dublin , ca ) . peptides were 
 separated on a 100 m  11 cm fused silica capillary column 
 ( polymicro technologies , llc . , phoenix , az ) and 100 m  
 6 cm fused silica capillary precolumn packed with 5 m , 300 
  jupiter c18 ( phenomenex , torrance , ca ) . liquid chromatography 
 was performed using a 95 min gradient at a flow rate of either 400 
 or 600 nl min using a gradient mixture of 0.1% 
 ( v / v ) formic acid in water ( solvent a ) and 0.1% ( v / v ) formic acid 
 in acetonitrile ( solvent b ) . briefly , a 15 min wash period ( 100% solvent 
 a ) was performed followed by a gradient to 98% a at 15 min ( 1.2 l 
 min ) , and eluent was diverted to waste prior to 
 the analytical column using a vented column set up similar to that 
 previously described . following removal 
 of residual salts , the flow was redirected to flow through the analytical 
 column and solvent b increased to 75% over 35 min and up to 90% in 
 65 min . the column was re - equlibrated to 98% solvent a for 10 min 
 after each run . all peptides were analyzed using targeted analysis 
 of doubly and/or triply charged ions to acquire the complete ms / ms 
 spectrum . ms analysis was performed on the neutral loss 
 of phosphoric acid for phosphopeptides iknlqpsldpsh 
 ( protea peptide ) , mhlppsptdsnyr , and gshqipsldnpdyqqdffpk ( both egfr phosphopeptides ) in addition 
 to ms / ms analysis . typical targeted parameters include an isolation 
 width of two bracketed around the m / z of interest , a fragmentation time of 10 ms , normalized collision 
 energy of 35.0 , spray voltage of 1.8 kv , and capillary temperature 
 at 200 c . data was analyzed using either xcalibur software 
 ( thermofisher , san jose , ca ) to determine extracted ion current peak 
 area for 35 transitions for each targeted peptide or the full 
 scan ms / ms filtering feature in skyline 1.1 software . each phosphopeptide was normalized by dividing the individual 
 phosphopeptide ( sum of three or four ion transitions / peptide ) by either 
 the individual reference peptides ( sum of three or four ion transitions / peptide ) 
 or the sum of all of the references ( unmodified internal peptides 
 from bsa or egfr ) peptides . full support for psrm using 
 chromatograms extracted from targeted ms / ms spectra for peak area 
 calculations was implemented in the skyline software tool , as shown 
 in figures 2 , 6 and 
 s1 ( supporting information ) , and released 
 in version 1.1 . these new features included method export for thermofisher 
 ltq instruments as well as chromatogram extraction from ms / ms spectra 
 at targeted product ion mass - to - charge ratios , making available for 
 psrm many existing skyline features proven in srm experiments with 
 triple quadrupole mass spectrometers . the relationship between response 
 and concentration was modeled by applying a weighted least - squares 
 with the robust linear model using tukey s biweight to down - weight 
 potential outliers . the model also accounts for nonlinear behavior at low concentrations 
 by incorporating change - points . selection of change points is based 
 on akaike s information criterion ( aic ) , where the optimal 
 model is the one with the minimum aic . the fitted model provided three 
 summary statistics : correlation coefficient ( r ) , slope , and coefficient of variation ( cv ) . this work describes 
 quantitative analysis of post - translationally modified ( ptm ) peptides 
 by psrm together with either stable isotope dilution ( sid ) or a new 
 irp method . the psrm experiments are targeted ms / ms analyses performed 
 by producing a full ms / ms spectrum for each precursor m / z in a target list using a linear ion trap mass 
 spectrometer ( ltq velos ) . transitions are extracted from the full 
 ms / ms or ms spectrum and peak areas for transitions are 
 summed and normalized to areas for a reference standard . for stable - isotope 
 dilution ( sid ) , the summed peak area is normalized to summed peak 
 areas for transitions from a stable isotope labeled peptide standard . 
 in the irp method , one or more unmodified proteotypic peptides from 
 the target protein serve as the reference standard for the modified 
 peptides in the analysis . because the target modified peptides and 
 the reference standard are present in the same protein , the irp method 
 corrects for variations in recovery of the protein in the analysis . normalized signals increase or decrease with a corresponding increase 
 or decrease in the stoichiometry of the modification . to test the ability of the irp method to detect differences in modification 
 stoichiometry as changes in normalized psrm ratio , we performed proof - of - principle 
 experiments by spiking synthetic phosphopeptides into a bsa background 
 ( see figure 1 ) . the peptides , drvpyihpf ( angiotensin ii ) and iknlqpsldpsh ( cholecystokinin 
 1020 ) , were spiked into 100 l of 6 g ml bsa digest at 0.01 , 0.02 , 0.05 , 0.10 , 0.20 , 0.50 , 
 1.0 , and 2.0 fmol ng bsa . these concentrations 
 were chosen to mimic the low abundance phosphorylation events that 
 occur in biological systems ; these spike 
 concentrations correlated to 0.1214% stoichiometry relative 
 to bsa . synthetic 
 phosphopeptides were spiked into a standard bovine serum albumin ( bsa ) 
 protein digest at increasing concentrations ( 0.06412.8 fmol 
 l ) . phosphorylated peptides were targeted 
 employing psrm using a linear ion - trap mass spectrometer . data is 
 normalized by dividing targeted phosphorylated peptide peak area ( sum 
 of three to four transitions ) by bsa peptide peak areas ( sum of three 
 transitions ) . response curves were generated 
 by analyzing 
 each concentration point ( five replicate lc ms injections ) 
 on an ltq velos linear ion trap using psrm . for each spiked peptide , extracted ion chromatograms ( xics ) for three to four transitions were 
 chosen based on stability ( low variability in peak area ) , sequence , 
 or modification - specific transitions ( when possible ) and strong transition 
 signals . figure 2 outlines the ms and product ion 
 filtering feature in skyline showing chromatographic profiles for 
 the high ( 2.0 fmol ng bsa ) and low ( 0.02 fmol 
 ng bsa ) concentration of phosphorylated peptide 
 spiked into bsa . this figure clearly demonstrates the advantage of 
 the psrm over ms signal extraction to quantify at lower 
 concentrations due to the increased specificity leading to a greater 
 signal - to - noise . we also acquired these data using an ltq - orbitrap 
 with the tandem ms collected on the ltq portion of the experiment 
 and the ms collected in the orbitrap portion of the instrument . the ms signal in the orbitrap was significantly improved 
 in the orbitrap and the psrm results were essentially the same ( data 
 not shown ) . the peptide sequences , precursor m / z and specific transitions extracted are listed in table 
 s41 ( supporting information ) . the sum of 
 transition peak areas for phosphopeptides was divided either by ( a ) 
 the sum of transition peak areas for individual reference bsa peptides 
 or by ( b ) the sum of the transition peak areas for all reference bsa 
 peptides . to assess the ability to use ms measurements 
 for quantification of protein modifications , ms of the 
 neutral loss ion [ m + 3h  h3po4 ] of phosphopeptide iknlqpsldpsh was also 
 measured . skyline display from replicate peak areas and imported targeted 
 full ms and ms / ms data from a high ( 2.0 fmol ng bsa ) , medium ( 0.2 fmol ng bsa ) , and low ( 0.02 
 fmol ng bsa ) concentration of a phosphorylated 
 peptide ( drvpyihpf ) spiked into bsa . samples were 
 run on a thermo fisher ltq - velos , low - resolution instrument . the precursor 
 ion , in blue , is filtered from ms scans taken at the beginning 
 of each cycle . at lower concentrations , interference becomes an issue 
 for the precursor in the ms scans ; however , the filtered 
 product ions from the targeted ms / ms remain selective and free from 
 interference producing a clear chromatographic peak . replicate peak 
 areas show the reproducibility of the peaks and their composition 
 from the fragment ion ( tandem ms ) traces . median cv across technical replicates plotted against 
 amount of 
 spiked in phosphorylated peptide standards . data point colors correspond 
 to reference peptide used for normalization . the median cvs decrease 
 as the amount of phosphopeptide spiked in background ( bsa digest ) 
 increases . ms / ms and ms data from five replicate 
 lc  ms 
 analyses of bsa digests spiked with both phosphopeptides were normalized 
 by the irp method and concentration response curves are shown in figure 
 s3 ( supporting information ) . concentration response 
 curve slopes , correlation coefficients ( r ) , and coefficients of variation ( cv ) for all reference peptides 
 are presented in table 1 . individual plots 
 for all of the measured peptides are shown in figures s5s7 
 ( supporting information ) and plots of the 
 median cvs for all replicates are in figure 3 . despite the fact that all of the normalization peptides were derived 
 from an equal amount of bsa in the sample , values for r , slope , and cv for normalization varied . the the utilization of the bsa reference 
 peptide yicdnqdtissk for quantation of the phosphorylated peptides 
 yielded the highest r values ( 0.96 ) , 
 lowest slope , and lowest median cv ( 10.8% ) compared to the 
 other reference peptides . similar values for r , slope , and cv were obtained for both ms / ms and ms measurements . the highest median cv was observed at the lowest phosphopeptide 
 spike amount ( 0.128 fmol ) for both phosphopeptides ( ms / ms and ms data ; figure 3 , supporting information ) . plots derived using single bsa reference peptides and using the 
 sum of all bsa reference peptides are presented in figures s8s10 
 ( supporting information ) . these experiments 
 were performed on a linear ion trap with automatic gain control ( agc ) , 
 which limits filling of the trap at higher ion currents , thus potentially 
 limiting linear dynamic range . in these studies , irp - normalized signals 
 appeared linear over the 200-fold concentration range examined , which 
 suggests that agc has little impact on response under the conditions 
 of our analyses . to evaluate the performance of the 
 irp method for relative quantification of specific protein phosphorylation 
 sites , we examined the changes in phosphorylation levels in egfr regulated 
 by egf stimulation and inhibition . an egfr overexpressing human epithelial 
 carcinoma cell line , a431 , was harvested ( 1 ) prior to any cell treatment 
 ( proliferating cells ) , ( 2 ) after serum starvation overnight and stimulation 
 with egf , ( 3 ) after cotreatment with 10 g ml cetuximab ( monoclonal antibody inhibitor of egfr ) and egf , and ( 4 ) 
 after cotreatment with 500 nm gefitinib ( small molecule egfr tyrosine 
 kinase inhibitor ) and egf . immunoblot analyses were performed 
 pre- and post ip for each treatment group for total egfr and specific py sites y998 and y1172 . egfr specific py sites y998 and 
 y1172 were chosen for immunoblot analysis because both sites have 
 high - quality , commercially available site - specific antibodies . phosphorylation 
 at both residues has been linked to pertinent egfr biology with y998 
 representing a phosphorylation site implicated in receptor endocytosis 
 and y1172 representing a site of autophosphorylation . the purpose 
 of these studies was not to produce equal levels of inhibition but 
 to produce detectable differences in phosphorylation between stimulated 
 and inhibited states using at least two known inhibitors of egfr . the amount of receptor phosphorylation at sites y998 and y1172 in 
 a431 cells ( figure 5 ) was significantly elevated 
 in egf treated samples over proliferating controls . as expected , both 
 cetuximab and gefitinib inhibitor treatments decreased the amount 
 of tyrosine phosphorylation detected when compared to egf treatment 
 alone . the egfr tyrosine kinase inhibitor gefitinib at 500 nm decreased 
 tyrosine phosphorylation below basal levels at both sites , whereas 
 at a dose of 10 g ml , cetuximab was not 
 as effective at inhibiting egfr phosphorylation . for quantitative 
 lc ms / ms experiments , egfr was immunoprecipitated 
 from the a431 cells with cetuximab in the presence of protease and 
 phosphatase inhibitors , separated on an sds - page gel , and the egfr 
 band was excised and digested in - gel with trypsin ( figure 4 ) . modified peptides corresponding 
 to phosphorylation at sites y998 ( mhlpsptdsnfpyr ) , 
 y1110 ( rpagsvqnpvpyhnqplnpapsr ) , y1172 ( gshqisldnpdpyqqdffpk ) , y1197 ( gstaenaepylr ) , s991 
 ( mhlppsptdsnfyr ) , and s1166 ( gshqipsldnpdyqqdffpk ) within egfr were targeted for quantification using 
 psrm . figure 6 shows the extracted ion chromatograms 
 using skyline for y1197 for each treatment type . phosphoserine sites 
 ( s991 and s1166 ) also were monitored using ms of the 98 
 da neutral loss ion corresponding to the neutral loss of phosphoric 
 acid . stable isotope - labeled peptides corresponding to sites y998 , 
 y1110 , y1172 , and y1197 were spiked in prior to psrm analysis to permit 
 comparison of the irp and sid methods for quantitation . five unmodified 
 peptides from egfr were selected for normalization using the irp method . the location and domain position ( extracellular , juxtamembrane , tyrosine 
 kinase or cytoplasmic ) of each reference and phosphorylated peptide 
 in egfr is presented in figure s11 ( supporting 
 information ) . internal reference peptides were selected based 
 on high signal stability and a wide range of elution times ( see figure 
 s12 , supporting information ) . for each 
 targeted peptide monitored , xics were selected for three to five transitions 
 that indicate modification specificity ( when possible ) and that generated 
 strong psrm transitions signals . although modification site - specific 
 transitions do not always produce sufficient signal for quantitation , 
 they frequently can be used to verify the site of modification . in 
 the case of y998 vs s991 phosphorylation , we were able to verify the 
 site not only with modification site - specific fragment ions but also 
 with the comparison to the y998 phosphorylated synthetic peptide . 
 extracted ion chromatograms for peptides y998 and s991 ( mhlpsptdsnfpyr and mhlppsptdsnfyr , respectively ; shown 
 in figure s2 , supporting information ) demonstrate 
 baseline separation of these species under our experimental conditions . since only one or two modification site - specific transitions were 
 detected , these ions were used primarily to distinguish the two distinct 
 phosphorylated peptide signals . it is possible 
 that the digestion of this peptide may be hindered when the tyrosine 
 is phosphorylated ( given its proximity to the tryptic cleavage site ) ; 
 we assumed that the relative amounts of both peptides ( mhlpsptdsnfpyr and mhlppsptdsnfyr ) with respect to 
 treatment should remain unaffected and digestion should be reproducible , 
 thus the conclusion is the same . we believe that this assumption is 
 reasonable since we are not comparing the absolute amount of these 
 two peptides and our data was consistent among the three biological 
 replicates . the absolute amount of the y998 peptide estimated by sid , 
 however , may be affected by this potential for missed cleavage . the 
 peptide sequences , precursor m / z , and specific transitions are listed in table s13 , supporting information . peak areas were calculated from the 
 sum of transitions for the phosphopeptides and then divided by the 
 peak areas for egfr internal reference peptides ( for irp ) or its stable 
 isotope - labeled standards ( for sid ) . three biological replicates were 
 performed for each treatment group and three lc  psrm ms 
 injections for each sample . ( a ) illustration of the immunoprecipitation 
 ( ip ) method for egfr 
 and treatment groups utilized in these experiments . after ip , the 
 egfr corresponding band on sds page gel was excised and targeted analysis 
 performed on an ltq velos mass spectrometer . ( b ) representation of 
 the analytical approach for normalizing six egfr phosphorylated peptides 
 to internal reference peptides ( irps ) . irps are unmodified peptides 
 within the protein of interest , in these experiments , egfr . a431 cells untreated , modulated with egf , or cotreated 
 with inhibitor 
 ( cetuximab or gefitinib ) followed by egf exhibit different egfr activation 
 statuses . immunoblot showing egfr activation prior to and after treatment(s ) 
 ( phosphorylation at y998 and y1172 ) . both phosphorylated forms of 
 the receptor were targeted in the psrm - ms method . input lane is 5% 
 of total protein load , and ip lane is post - immunoprecipitation . skyline display from replicate peak areas and imported 
 targeted 
 ms / ms data to produce psrm traces for gstaenaepylr 
 from a431 cells not treated ( proliferating ) , modulated with egf , or 
 cotreated with inhibitor ( cetuximab or gefitinib ) followed by egf . replicate peak 
 areas show the reproducibility of the peaks and their composition 
 from the psrm traces . to evaluate the performance of the irp method , 
 we compared cv values 
 for measurements using different irp peptides together with sid analyses 
 for each of the six egfr phosphorylation sites analyzed ( table 2 ) . plots of cvs for technical replicate analyses 
 from each of three biological replicate experiments are presented 
 in figures s14s19 ( supporting information ) . normalization to the egfr peptide 
 iplenlqiir yielded the lowest global cv ( median 22% ) , whereas the 
 other normalization peptides nlqeilhgavr ( median 27% ) , eisdgdviisgnk 
 ( median 27% ) , glwipegek ( median 26% ) , and itdfglak ( median 32% ) displayed 
 modestly greater variation . cv values differed between distinct biological 
 replicate experiments . in our studies , biological replicate experiments 
 1 and 2 displayed lower variability , with median cvs from different 
 irp peptides averaging approximately 30% , whereas biological replicate 
 experiment 3 yielded more variation . this higher variation was caused 
 by individual technical replicate runs ( individual injections on the 
 mass spectrometer using the same sample ) , some of which produced low 
 signal across the chromatogram . because these psrm analyses employed 
 ion trap analyzers , we were able to compare ion injection times for 
 low - signal versus high - signal runs . technical replicate runs with 
 low - signal displayed considerably longer ion injection times and correspondingly 
 low signal for summed mrm transitions ( compare figures s2021 
 ( low - signal run ) with figures s2223 ( high - signal run ) , supporting information . this phenomenon was most 
 likely due to instrumental issue , such as an incomplete injection 
 or some other signal failure in the mass spectrometer . in all of the 
 experiments , the sid method yielded significantly greater measurement 
 precision with a global median cv of 15% . the data generated by analyses of immunoprecipitated 
 egfr from 
 biological experiment 1 by the irp and sid methods are shown in figure 7 . both methods yielded similar measures of phosphorylation 
 at y1172 ( gshqisldnpdpyqqdffpk ) , its stimulation 
 by egf , and inhibition by cetuximab and gefitinib ( figure 7a , b ) . the irp method produced similar results to 
 sid for all four phosphotyrosine sites ( y998 , y1110 , y1172 , and y1197 ) 
 ( figure 7c and figures s24s27 , supporting information ) . these results are also 
 consistent with the immunoblot analysis for y1172 shown in figure 5 . egf - treated stimulation produced the highest normalized 
 psrm signal , whereas samples cotreated with 500 nm gefitinib showed 
 profound decreases in y1172 phosphorylation to below basal ( proliferating ) 
 levels . co - treatment with cetuximab produced less inhibition in y1172 
 phosphorylation to near basal levels . figure 7c represents the degree of site - specific phosphorylation relative 
 to that for egf stimulation by a phosphorylation index , which was 
 calculated as the ratio of the proliferating ( p ) , gefitinib ( g+e ) , 
 or cetuximab ( c+e ) normalized psrm signal to the egf stimulated normalized 
 psrm signal ( eq 1 ) . although we monitored the 
 corresponding peptides without phosphorylation ( data not shown ) , we 
 did not see any appreciable changes in the quantitation of these peptides , 
 most likely due to the low stoichiometry of the phosphorylation ( data 
 not shown).1 treatment groups show 
 the same trends when py peptides 
 are normalized to an internal reference peptide or its stable isotope 
 labeled counterpart . egfr peptide py1172 ( gshqisldnpdpyqqdffpk ) normalized to an ( a ) internal reference peptide 
 ( iplenlqiir ) and ( b ) its py sid peptide standard . ( c ) phosphorylation index for each egfr py peptide 
 and cell treatment group . similar trends are observed for each py targeted peptide after normalization to an irp ( red ) 
 or its stable isotope labeled counterpart ( purple ) . these data represent 
 three technical lc  psrm  ms injections of biological 
 replicate 1 . the underlined amino acid indicates which amino acid was stable isotope - labeled . 
 for biological replicate 1 , cv values ranged between 9.847% 
 and 7.332% for the irp and sid method , respectively . the higher 
 cv values were generated from geftinib + egf samples , and the median 
 cv values ranged were 21% and 24% , respectively . both irp ( iplenlqiir ) and sid methods yielded similar 
 results and 
 consistently detected similar phosphorylation status differences among 
 the four treatment groups . for ps and pt peptides whose ms / ms spectra are dominated by neutral loss of h3po4 , measurements based on ms fragmentation 
 of the neutral loss ion may offer higher confidence sequence - specific 
 detection . ms measurements for relative quantification 
 of phosphoserine modifications were performed on peptides for egfr 
 sites s991 ( mhlppsptdsnfyr ) [ m + 2h  h3po4 ] and s1166 ( gshqipsldnpdyqqdffpk ) [ m + 3h  h3po4 ] . peak areas from ms measurements were normalized 
 to ms / ms - derived peak areas for the 5 egfr irp sequences described 
 above , as well as to the ms / ms - derived peak areas for the isotope - labeled py peptide standards used for sid analyses of the py forms of these sequences ( see above ) . median cv plots 
 using the irp method for s991 and s1166 phosphopeptides ( figures s18 
 and s19 , supporting information ) indicate 
 that normalization to the iplenlqiir peptide produced the smallest 
 measurement variation , which was comparable to that achieved with 
 normalization to the synthetic py sid peptide sequence 
 analogs . both analysis methods yielded similar estimates of phosphorylation 
 changes induced by egf and the effects of cetuximab and gefitinib 
 ( figure 8 and figures s28 and s29 , supporting information ) . phosphopeptide ps991 ( mhlppsptdsnfyr ) normalized to an ( a ) irp or ( b ) py sid peptide standard ( mhlpsptdsnfpyr ) shows similar trends . phosphopeptide ps1166 ( gshqipsldnpdyqqdffpk ) normalized 
 to an ( c ) irp or ( d ) py sid peptide standard ( gshqisldnpdpyqqdffpk ) show similar trends . these 
 data show that ms measurements can be used for quantification 
 of protein modifications . phosphorylation levels for s991 display a pattern 
 similar to that 
 for the four phosphotyrosines described above . phosphorylation at 
 s991 increased after egf treatment compared to basal ( proliferating ) 
 levels and were reversed , although not completely , by both inhibitors . this result differs somewhat from the near complete reversal of phosphotyrosine 
 modification we observed above for gefitinib . similar results for 
 s991 phosphorylation were reported by stover et al . , who used mass spectrometry analyses to detect s991 phosphorylation 
 induced by egf stimulation and inhibited by the egfr inhibitor pk166 . in contrast , similar analyses of phosphorylation at s1166 ( gshqipsldnpdyqqdffpk ) using both methods indicated a very different 
 pattern . egf treatment produced little or no s1166 phosphorylation , 
 although gefitinib further decreased phosphorylation at this site . the lack of significant egf - induced phosphorylation at s1166 is consistent 
 with a previous study that employed a mass spectrometry method . on the other hand , our major goal in this work was to evaluate 
 the irp method for 
 quantifying changes in protein ptm and to demonstrate the utility 
 of psrm for quantification . although we have previously reported a 
 similar approach , we describe here the performance 
 characteristics of the method in comparison to sid . we further describe 
 the implementation of ms / ms and ms - based psrm measurements 
 on an ltq velos ion trap instrument . the psrm transitions can be extracted 
 from ms / ms and ms data , normalized to peak areas from 
 reference peptides within the same protein , thus affording relative 
 quantification of protein modifications . unlike mrm data , psrm data 
 records a full ms / ms spectrum for each monitored peptide , which allows 
 for spectrum verification by visual inspection as well as the ability 
 to choose different ions to extract based on ptm site ( e.g. , ps , pt vs py ) ( see figure 2 and figures s1 and s2 , supporting 
 information ) . as with other ms - based methods , the irp method 
 can measure multiple site - specific phosphorylation sites in parallel 
 without the need for site - specific antibodies and without potential 
 concerns about antibody cross - reactivity and lack of phospho - site 
 specificity . although the method lacks the analytical precision of 
 sid , the needless requirement for labeled internal standards , the 
 ease of implementation and the suitability for typical quantitative 
 comparisons , as illustrated with our analyses of egfr phosphorylation , 
 make this method suitable for broad application in protein biochemistry . previous label - free quantitation approaches have utilized estimated 
 stoichiometry ( es ) , flyability ratios , the native reference peptide 
 ( nrp ) method or the selected ion tracing method to quantify post - translational 
 modifications . in all of these methods , xics ( ion currents ) at the ms level are generated for each site specific modification as well 
 as its unmodified peptide complement or an unmodified reference peptide . these methods calculate the stoichiometry ( or site abundance ) of individual 
 post - translationally modified sites by taking the modified peptide 
 peak area and normalizing to the sum of modified and unmodified peak 
 area , to an unmodified reference peptide 
 peak area or to the sum of unmodified peak area plus the peak 
 area of any other possible sites of modification on the target peptide . a key difference between our approach and previous 
 methods is the use of ms / ms extracted ion chromatograms rather than 
 ms data for each modified and reference peptide . unlike 
 the es method and other ms - based methods , the psrm utilizes 
 ms / ms and ms data to obtain peptide sequence and site 
 specific localization data , thus allowing not only verification of 
 the site of modification but also site - specific quantitation even 
 when peptide peaks can not be resolved ( see figure 2 and figure s1 , supporting information ) . our initial proof - of - principle experiment analyzed two phosphopeptides 
 spiked at increasing concentrations into a bsa digest . the simulated 
 irp method by psrm achieved a linear response on the ltq velos ion - trap 
 mass spectrometer across 2 orders of magnitude for all normalization 
 peptides examined ( figure s3 , supporting information ) . some normalizing peptides produced a more consistent response , 
 which correlated to lower variability ( < 10% cv , see table 1 ) . although the higher end of the standard curve 
 may exceed the concentration of modified peptide that would typically 
 occur in a protein , we examined higher concentrations to determine 
 whether the automatic gain control ( agc ) affected linearity of response . 
 even at higher concentrations , where ion injection times were shortened 
 by agc ( to limit the ions according to the set target intensity value ) , the detected response remained linear ( see r values in table 1 ) using these instrument 
 settings . as expected , lower phosphopeptide concentrations yielded 
 greater measurement variability , as indicated by higher median cv 
 measurements ( > 30% median cv for 0.13 fmol ( 0.01 fmol ng of bsa ) phosphopeptide ( figure 3 ) . higher 
 cv values are typical for peptide concentrations at or near the limit 
 of detection due to the decreased signal - to - noise that is typically 
 observed . it should be noted in these types of experiments there needs 
 to be a balance between the approapriate maximum target intensity 
 value and the ion injection time settings for agc . we observed linearity 
 in our proof - of - principle experiment despite a nearly 1000-fold difference 
 between the reference peptide and modified peptide concentrations 
 ( lowest point modeled 0.12% stoichiometry ) . large differences in signal 
 intensities of the reference peptides and modified peptides may still 
 create nonlinearity and space charging in ion traps may also affect 
 quantitation , so this should be considered in instrument acquisition 
 settings . median cvs showed lower variations ( 20% ) as phosphopeptide 
 analyte concentration increased from 0.13 to 25.6 fmol ( 0.01 fmol 
 ng of bsa up to 2.00 fmol ng of bsa ) ( figure 3 ) . elevated cvs for both 
 sid and irp methods also were obtained in gefitinib- and egf - treated 
 samples , which typically had low levels of phosphorylated peptides 
 and where signals approached the limit of detection ( figures s14s19 , supporting information ) . in the bsa spike 
 model , standard curves for the ps peptide iknlqpsldpsh ( ms data ) had r values , slopes , and cvs similar to or better 
 than those obtained from ms / ms data when using the same normalizing 
 peptide ( table 1 ) . although ion intensity for 
 the ms spectra were approximately 10-fold lower than the 
 ms / ms data , the ms data typically had better signal - to - noise 
 ratios and thus similar or better cvs . ms data thus can 
 be used for relative quantification of protein modifications , especially 
 when ms / ms data of ps or pt containing 
 peptides do not provide adequate fragment ion data for site - specific 
 modification mapping or sequence determination . higher order tandem 
 ms experiments such as the ms measurements demonstrated 
 here can not be obtained in quadrupole mass analyzers traditionally 
 used for quantification , but can be performed on ion trap instruments . in the egfr phosphorylation studies , internal reference peptides 
 were chosen such that ( 1 ) they were known to be unmodified , ( 2 ) eluted 
 across the chromatogram , ( 3 ) displayed consistent signal stability , 
 ( 4 ) were observed in previous data dependent lc - ms / ms data , and ( 5 ) 
 contained between 7 and 20 amino acids and preferably lacked methionine 
 and cysteine residues . in these studies , we did not identify any other 
 measures ( e.g. , location in lc elution time , similarity in peptide 
 length / sequence , etc . ) to predict the best irp to use . phosphopeptides 
 were normalized individually to the each of the five internal reference 
 peptides ( itdfglak , iplenlqiir , glwipegek , nlqeilhgavr , and eisdgdviisgnk ) . individual analyses of the internal reference peptides indicated that 
 some internal reference peptides showed large differences in the range 
 and median cv plots ( figures s14s19 , supporting 
 information ) . our results show that multiple irps should be 
 evaluated to optimize the performance of the irp method and to ensure 
 that any given reference peptide does not conflict with the results 
 of the others . ultimately , we chose the internal reference peptide 
 with the lowest median cv ( highest signal stability ) in the data set , 
 unless there were clear contradictory results to the other reference 
 peptides ( as was the case for the glwipegek reference peptide described 
 below ) . we studied biological variations of egfr phosphorylation 
 in a431 
 cells under four treatment conditions . analyses with the irp method 
 using all internal reference peptides ( except glwipegek ) detected 
 the same differences between treatments as did sid analyses . this 
 demonstrates that moderate differences in measurement variation do 
 not significantly impact the biological conclusions drawn from these 
 studies . in our sid analysis of 4 analyzed phosphotyrosine sites in 
 egfr , median cvs ranged from 5 to 15% , whereas analyses with the irp 
 method ( using reference peptide iplenlqiir ) of the same four sites 
 had median cvs around 20% ( figures s14s19 , supporting information ) . although the median cv for the irp 
 method was greater than the sid method , the interpretation of phosphorylation 
 differences with both methods is the same . we also monitored phosphorylation 
 sites y998 and y1172 with commercially available antibodies and these 
 analyses confirmed the mass spectrometry results ( figure 5 ) . by all three methods ( immunoblot , sid and irp ) , gefitinib 
 was a more potent inhibitor of egfr than cetuximab at indicated concentrations ; 
 immunoblot analyses for y998 and y1172 were barely above background 
 and both sid and irp methods for sites y998 , y1110 , y1172 , and y1197 
 were calculated to have a phosphorylation index < 10% when compared 
 to egf treated cells . the corresponding nonphosphorylated peptides 
 did not appear to significantly change in response to treatment . this 
 is most likely due to the very low stoichiometry of these phosphorylations , 
 consistent with previous studies . analysis using ms spectra for two ps containing peptides in egfr , mhlppsptdsnfyr ( s991 ) , 
 and gshqipsldnpdyqqdffpk ( s1166 ) yielded results 
 consistent with previous literature reports . phosphoserine peptide s991 follows the same overall changes as the 
 four egfr py sites ( y998 , y1110 , y1172 , and y1197 ) ; 
 with the exception that gefitinib was not a more potent inhibitor 
 than cetuximab ( i.e. , at the concentrations used gefitinib and cetuximab 
 have similar inhibitory effects on s991 ) . trends across the different 
 treatment groups were consistent whether the msps peptides were normalized to the isotope - labeled py sid peptide standard or to the internal reference peptide 
 with the lowest median cv ( iplenlqiir ) . the effect of egfr inhibitors 
 on phosphorylation at site s1166 and site s991 has not previously 
 been reported . in these experiments , the sid and irp methods 
 were analyzed on 
 a linear ion trap mass spectrometer ( ltq velos ) , which can monitor 
 20 peptides in a single lc while psrm measurements do not provide the same throughput as triple 
 quadrupole based mrm analyses ( > 30 peptides can be measured with 
 four 
 transitions in a single , unscheduled lc triple quadrupole mrm 
 analysis ) , the added benefit of the peptide sequence confirmation 
 from the full ms / ms spectrum and the ability to acquire higher order 
 tandem ms data are significant advantages . the psrm approach also 
 allows selection of the transition after ms / ms analysis as well as 
 quantitation using fragment ions when ms data is poor 
 ( see figure 2 ) . having the tandem ms spectrum 
 enables quantitative extension of modification mapping experiments 
 without transferring methods to another platform . our results suggest 
 several precautions that can improve the reliability of psrm analyses . 
 first , multiple technical replicate injections enable assessment of 
 instrument performance - based variation due to chromatography , detectors , 
 ion injection times , and signal intensity . second , an irp method should 
 incorporate multiple internal reference peptides to provide confirmatory 
 results , to identify peptides with the lowest variation and to minimize 
 error due to ion suppression effects , and coeluting interferences . 
 in our studies , we observed a consistently poor performing internal 
 reference peptide , glwipegek , which generated large variations in 
 cv for normalized psrm signal ( figures s14s19 , supporting information ) and also generated egf- 
 and inhibitor - related phosphorylation differences inconsistent with 
 analyses using the other internal reference peptides ( i.e. , s1166 , 
 see figure s29 , supporting information ) . the glwipegek internal reference peptide may perform poorly due to 
 inconsistent digestion resulting in a c - terminal missed cleavage ( see 
 figure s11 , supporting information ) or 
 due to some interfering ions of similar m / z adding to the noise of the signal or causing signal suppression . if the inconsistency is due to similar m / z adding to the noise , this could potentially be resolved 
 using a higher resolution instrument for this approach . arbitrary 
 selection of a single reference peptide would not have detected this 
 poor perfoming peptide . theoretical prediction of optimal internal 
 reference peptides may not be adequate and may require the examination 
 of multiple internal reference peptide signals in a biological matrix 
 to ensure consistency . we employ the irp method for analyses 
 of modifications on individual 
 proteins in relatively simple samples , such as immunoprecipitated 
 proteins or proteins isolated from sds  page gel bands . we have 
 not considered and do not recommend the irp method for global analyses 
 of modified proteins in complex proteomes . in the appropriate context , 
 the irp method is intended to estimate differences in protein modifications 
 between similar samples . the data are comparable in measurement variation 
 to immunoblot analyses ( cvs up to 40% ) . ms - based analyses , such as 
 the irp and sid methods , are able to selectively measure many site - specific 
 changes for which reliable antibody reagents are unavailable . the 
 irp method displays lower precision than sid , but it is useful in 
 many applications where high precision is not required . irp - based 
 methods also do not use labeled peptide standards , which significantly 
 increases analysis costs . in the context of targeted biochemical analyses , 
 the irp method accounts for variations in immunoprecipitation or affinity 
 capture efficiency and gel fractionation . by utilizing multiple peptides 
 for normalization , we account for variable digestion or other unknown 
 modification of the normalization peptides . these 
 features make the irp method a flexible , general approach for comparative 
 analysis of protein modifications which can find widespread application 
 in biochemical analyses .
liquid chromatography tandem mass spectrometry ( lc  ms / ms ) 
 based methods provide powerful tools for the quantitative analysis 
 of modified proteins . we have developed a label - free approach using 
 internal reference peptides ( irp ) from the target protein for signal 
 normalization without the need for isotope labeling . 
 ion - trap mass 
 spectrometry and pseudo - selected reaction monitoring ( psrm ) were used 
 to acquire full ms / ms and ms3 spectra from target peptides . 
 skyline , a widely used software for srm experiments , was used for 
 chromatographic ion extraction . 
 phosphopeptides spiked into a bsa 
 background yielded concentration response curves with high correlation 
 coefficients ( typically > 0.9 ) and low coefficients of variation 
 ( 15% ) 
 over a 200-fold concentration range . 
 stable isotope dilution ( sid ) 
 and irp methods were compared for quantitation of six site - specific 
 phosphorylations in the epidermal growth factor receptor ( egfr ) in 
 epidermal growth factor - stimulated a431 cells with or without the 
 addition of egfr inhibitors cetuximab and gefitinib . 
 equivalent responses 
 were observed with both irp and sid methods , although analyses using 
 the irp method typically had higher median cvs ( 2231% ) than 
 sid ( 1020% ) . 
 analyses using both methods were consistent with 
 immunoblot using site - selective antibodies . 
 the ease of implementation 
 and the suitability for targeted quantitative comparisons make this 
 method suitable for broad application in protein biochemistry .