Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. The analysis of complex mixtures by shotgun proteomics provides a snapshot of the protein components of the cell under various cellular conditions. Many proteomics studies have focused on the characterization of the protein content from highly complex mixtures such as nuclear extract from diverse organisms. Although these studies provide a wealth of information, the analysis of purified protein complexes has the potential to not only identify differential protein expression, but also to identify differentially interacting proteins and post-translational modifications that are present at undetectable levels in large-scale analyses. There have been several studies that analyze protein complexes using quantitative proteomic methods, however many of these studies have relied on binary quantitative values (presence or absence) rather than using methods that rely on discrete values (1.Krogan N.J. Cagney G. Yu H. Zhong G. Guo X. Ignatchenko A. Li J. Pu S. Datta N. Tikuisis A.P. Punna T. Peregrin-Alvarez J.M. Shales M. Zhang X. Davey M. Robinson M.D. Paccanaro A. Bray J.E. Sheung A. Beattie B. Richards D.P. Canadien V. Lalev A. Mena F. Wong P. Starostine A. Canete M.M. Vlasblom J. Wu S. Orsi C. Collins S.R. Chandran S. Haw R. Rilstone J.J. Gandi K. Thompson N.J. Musso G. St Onge P. Ghanny S. Lam M.H. Butland G. Altaf-Ul A.M. Kanaya S. Shilatifard A. O'Shea E. Weissman J.S. Ingles C.J. Hughes T.R. Parkinson J. Gerstein M. Wodak S.J. Emili A. Greenblatt J.F. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.Nature. 2006; 440: 637-643Crossref PubMed Scopus (2316) Google Scholar, 2.Gavin A.C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Höfert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Functional organization of the yeast proteome by systematic analysis of protein complexes.Nature. 2002; 415: 141-147Crossref PubMed Scopus (3972) Google Scholar, 3.Collins S.R. Kemmeren P. Zhao X.C. Greenblatt J.F. Spencer F. Holstege F.C. Weissman J.S. Krogan N.J. Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae.Mol. Cell Proteomics. 2007; 6: 439-450Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). In this study, we address the reproducibility of label-free spectral count-based proteomics by analyzing biological replicate purifications of RNA polymerase I, II, and III.The transcription of cellular RNAs is performed by large multiprotein enzyme complexes known as the RNA polymerases (RNAPs) 1The abbreviations used are:RNAPRNA polymeraseMuPITmultidimensional protein identification technologyrRNAribosomal RNAmRNAmitochondrial RNAtRNAtransfer RNAdSNAFdistributed normalized spectral abundance factordSIFDRB sensitivity inducing factorTFIIFtranscription factor II FLTQlinear trap quadrupoleAPEXabsolute protein expressionGOGene Ontology. 1The abbreviations used are:RNAPRNA polymeraseMuPITmultidimensional protein identification technologyrRNAribosomal RNAmRNAmitochondrial RNAtRNAtransfer RNAdSNAFdistributed normalized spectral abundance factordSIFDRB sensitivity inducing factorTFIIFtranscription factor II FLTQlinear trap quadrupoleAPEXabsolute protein expressionGOGene Ontology.. Yeast and other higher eukaryotes have three RNA polymerases known as RNA polymerase I, II, and III that are mainly responsible for the transcription of ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA), respectively. Each complex has a unique subunit composition with RNAPII containing 12 subunits, RNAPI containing 14 subunits, and RNAPIII containing 17 subunits; which is thought to contribute to their different target specificities. Five RNAP subunits, Rpb5, 6, 8, 10, and 12 are found in all three enzymes (for review, see (4.Cramer P. Armache K.J. Baumli S. Benkert S. Brueckner F. Buchen C. Damsma G.E. Dengl S. Geiger S.R. Jasiak A.J. Jawhari A. Jennebach S. Kamenski T. Kettenberger H. Kuhn C.D. Lehmann E. Leike K. Sydow J.F. Vannini A. Structure of eukaryotic RNA polymerases.Annu. Rev. Biophys. 2008; 37: 337-352Crossref PubMed Scopus (211) Google Scholar)). Structural studies on the three enzymes have yielded a great deal of information on their function, with the most insight coming from x-ray crystal structures on both a 10- and 12-subunit RNAPII, both in the presence and absence of DNA (4.Cramer P. Armache K.J. Baumli S. Benkert S. Brueckner F. Buchen C. Damsma G.E. Dengl S. Geiger S.R. Jasiak A.J. Jawhari A. Jennebach S. Kamenski T. Kettenberger H. Kuhn C.D. Lehmann E. Leike K. Sydow J.F. Vannini A. Structure of eukaryotic RNA polymerases.Annu. Rev. Biophys. 2008; 37: 337-352Crossref PubMed Scopus (211) Google Scholar, 5.Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.Science. 2001; 292: 1876-1882Crossref PubMed Scopus (741) Google Scholar, 6.Cramer P. Bushnell D.A. Kornberg R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science. 2001; 292: 1863-1876Crossref PubMed Scopus (953) Google Scholar). In addition, cryo-EM structures of the 14-subunit RNAPI complex and the 17-subunit RNAPIII complex have also been reported (7.Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Functional architecture of RNA polymerase I.Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 8.Fernández-Tornero C. Böttcher B. Riva M. C. A. G. transcription and from the of yeast RNA polymerase 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). studies provide a for quantitation of RNA polymerase I, II, and III all three enzymes have been reported to with a all subunits, information that is not for most multiprotein complexes A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.Science. 2001; 292: 1876-1882Crossref PubMed Scopus (741) Google Scholar, C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Functional architecture of RNA polymerase I.Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, K.J. S. A. Cramer P. of RNA polymerase II and Full Text Full Text PDF PubMed Scopus Google Scholar, K. Vannini A. Cramer P. A.J. Structural of RNA polymerase 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). of using these complexes is that the RNA polymerases are of from to in an for of the quantitation of both large and protein interact with a of low level associated proteins that transcription and RNA in to detection and quantitation for interacting this study, we have performed purifications of the RNA polymerases from Saccharomyces and the proteins using multidimensional protein identification technology using label-free quantitative proteomics methods, we have the reproducibility of the quantitation using spectral We show that quantitation using the normalized spectral abundance factor or spectral counting is highly reproducible across biological replicates. enzyme a of and different with RNAP II in the RNAP to the and RNAP III to both the and we also to the in the associated proteins found in we found that complex is associated with of proteins that a in such diverse as elongation and ribosomal RNA We focused on the analysis of highly enriched complexes found in with RNA polymerase II, sensitivity inducing factor complex and transcription factor II data show that label-free quantitative proteomic analysis of protein complexes highly reproducible peptide level quantitation as well as in-depth information on associated proteins that provide a snapshot of RNA polymerase protein have that quantitation using label-free spectral counting is a highly reproducible at both the protein and peptide unique are differentially by the proteins have a higher of than other We have that this is for the of RNAPII with a higher of spectral unique protein on the of protein complex in the same such as the use of at known A.C. C. S. D. proteomic analysis of complexes using normalized spectral abundance 2006; PubMed Scopus Google Scholar). The use of other quantitative methods such as quantitation also not able to for these sampling proteins with are not Although the ability to proteins a biological is a in the level of spectral sampling reproducibility biological supports across quantitation by the same peptide in when complexes are under different cellular the of a peptide to using a proteomics in spectral sampling levels for when protein abundance We to for the in the level of spectral sampling by using the APEX protocol as previously reported S. C. R. R.D. R. The APEX protein quantitation from proteomics 2008; PubMed Scopus Google Scholar, P. C. R. X. protein the of and 2007; PubMed Scopus Google Scholar, C. and protein abundance from protein 2008; PubMed Scopus Google Scholar). In addition, we used different of data from the analysis of lysate and from replicate of a highly purified and related of protein complexes. of was also not able to the in spectral sampling observed for the different RNA polymerase by the that the peptide detectability models are using binary values not and for peptide detection in the data The use of discrete values as of spectral the model based on the we and that replicate analyses. in peptide detectability for the same protein shows the of using discrete values for computational used for peptide For this we that the for label-free studies is the of advanced computational that are able to the of spectral peptide both the of the and of protein abundance Toward this the data from our analysis is as in the methods addition, we have that the use of biological replicate analysis using quantitative label-free proteomics provide insight the of low level interacting proteins such as the of the protein complexes DSIF and TFIIF. of DSIF and TFIIF have been to with RNAPII the as most by A. M. M. Leike K. J. Cramer P. of the RNA polymerase II transcription PubMed Scopus Google Scholar). We have that the proteins these complexes higher in their values across biological when with replicates. The supports the that proteins that interact with a purified complex in a dynamic fashion in higher in the quantitation across different biological replicates. is by the that was with a of reproducibility across all present at levels than DSIF and TFIIF of quantitative methods to the abundance of proteins a we able to quantitation of low level associated proteins and complex The analysis of complex mixtures by shotgun proteomics provides a snapshot of the protein components of the cell under various cellular conditions. Many proteomics studies have focused on the characterization of the protein content from highly complex mixtures such as nuclear extract from diverse organisms. Although these studies provide a wealth of information, the analysis of purified protein complexes has the potential to not only identify differential protein expression, but also to identify differentially interacting proteins and post-translational modifications that are present at undetectable levels in large-scale analyses. There have been several studies that analyze protein complexes using quantitative proteomic methods, however many of these studies have relied on binary quantitative values (presence or absence) rather than using methods that rely on discrete values (1.Krogan N.J. Cagney G. Yu H. Zhong G. Guo X. Ignatchenko A. Li J. Pu S. Datta N. Tikuisis A.P. Punna T. Peregrin-Alvarez J.M. Shales M. Zhang X. Davey M. Robinson M.D. Paccanaro A. Bray J.E. Sheung A. Beattie B. Richards D.P. Canadien V. Lalev A. Mena F. Wong P. Starostine A. Canete M.M. Vlasblom J. Wu S. Orsi C. Collins S.R. Chandran S. Haw R. Rilstone J.J. Gandi K. Thompson N.J. Musso G. St Onge P. Ghanny S. Lam M.H. Butland G. Altaf-Ul A.M. Kanaya S. Shilatifard A. O'Shea E. Weissman J.S. Ingles C.J. Hughes T.R. Parkinson J. Gerstein M. Wodak S.J. Emili A. Greenblatt J.F. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.Nature. 2006; 440: 637-643Crossref PubMed Scopus (2316) Google Scholar, 2.Gavin A.C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Höfert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Functional organization of the yeast proteome by systematic analysis of protein complexes.Nature. 2002; 415: 141-147Crossref PubMed Scopus (3972) Google Scholar, 3.Collins S.R. Kemmeren P. Zhao X.C. Greenblatt J.F. Spencer F. Holstege F.C. Weissman J.S. Krogan N.J. Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae.Mol. Cell Proteomics. 2007; 6: 439-450Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). In this study, we address the reproducibility of label-free spectral count-based proteomics by analyzing biological replicate purifications of RNA polymerase I, II, and III. The transcription of cellular RNAs is performed by large multiprotein enzyme complexes known as the RNA polymerases (RNAPs) 1The abbreviations used are:RNAPRNA polymeraseMuPITmultidimensional protein identification technologyrRNAribosomal RNAmRNAmitochondrial RNAtRNAtransfer RNAdSNAFdistributed normalized spectral abundance factordSIFDRB sensitivity inducing factorTFIIFtranscription factor II FLTQlinear trap quadrupoleAPEXabsolute protein expressionGOGene Ontology. 1The abbreviations used are:RNAPRNA polymeraseMuPITmultidimensional protein identification technologyrRNAribosomal RNAmRNAmitochondrial RNAtRNAtransfer RNAdSNAFdistributed normalized spectral abundance factordSIFDRB sensitivity inducing factorTFIIFtranscription factor II FLTQlinear trap quadrupoleAPEXabsolute protein expressionGOGene Ontology.. Yeast and other higher eukaryotes have three RNA polymerases known as RNA polymerase I, II, and III that are mainly responsible for the transcription of ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA), respectively. Each complex has a unique subunit composition with RNAPII containing 12 subunits, RNAPI containing 14 subunits, and RNAPIII containing 17 subunits; which is thought to contribute to their different target specificities. Five RNAP subunits, Rpb5, 6, 8, 10, and 12 are found in all three enzymes (for review, see (4.Cramer P. Armache K.J. Baumli S. Benkert S. Brueckner F. Buchen C. Damsma G.E. Dengl S. Geiger S.R. Jasiak A.J. Jawhari A. Jennebach S. Kamenski T. Kettenberger H. Kuhn C.D. Lehmann E. Leike K. Sydow J.F. Vannini A. Structure of eukaryotic RNA polymerases.Annu. Rev. Biophys. 2008; 37: 337-352Crossref PubMed Scopus (211) Google Scholar)). Structural studies on the three enzymes have yielded a great deal of information on their function, with the most insight coming from x-ray crystal structures on both a 10- and 12-subunit RNAPII, both in the presence and absence of DNA (4.Cramer P. Armache K.J. Baumli S. Benkert S. Brueckner F. Buchen C. Damsma G.E. Dengl S. Geiger S.R. Jasiak A.J. Jawhari A. Jennebach S. Kamenski T. Kettenberger H. Kuhn C.D. Lehmann E. Leike K. Sydow J.F. Vannini A. Structure of eukaryotic RNA polymerases.Annu. Rev. Biophys. 2008; 37: 337-352Crossref PubMed Scopus (211) Google Scholar, 5.Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.Science. 2001; 292: 1876-1882Crossref PubMed Scopus (741) Google Scholar, 6.Cramer P. Bushnell D.A. Kornberg R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science. 2001; 292: 1863-1876Crossref PubMed Scopus (953) Google Scholar). In addition, cryo-EM structures of the 14-subunit RNAPI complex and the 17-subunit RNAPIII complex have also been reported (7.Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Functional architecture of RNA polymerase I.Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 8.Fernández-Tornero C. Böttcher B. Riva M. C. A. G. transcription and from the of yeast RNA polymerase 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). studies provide a for quantitation of RNA polymerase I, II, and III all three enzymes have been reported to with a all subunits, information that is not for most multiprotein complexes A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.Science. 2001; 292: 1876-1882Crossref PubMed Scopus (741) Google Scholar, C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Functional architecture of RNA polymerase I.Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, K.J. S. A. Cramer P. of RNA polymerase II and Full Text Full Text PDF PubMed Scopus Google Scholar, K. Vannini A. Cramer P. A.J. Structural of RNA polymerase 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). of using these complexes is that the RNA polymerases are of from to in an for of the quantitation of both large and protein interact with a of low level associated proteins that transcription and RNA in to detection and quantitation for interacting RNA polymerase multidimensional protein identification technology ribosomal RNA RNA transfer RNA normalized spectral abundance factor sensitivity inducing factor transcription factor II trap protein Ontology. RNA polymerase multidimensional protein identification technology ribosomal RNA RNA transfer RNA normalized spectral abundance factor sensitivity inducing factor transcription factor II trap protein Ontology. In this study, we have performed purifications of the RNA polymerases from Saccharomyces and the proteins using multidimensional protein identification technology using label-free quantitative proteomics methods, we have the reproducibility of the quantitation using spectral We show that quantitation using the normalized spectral abundance factor or spectral counting is highly reproducible across biological replicates. enzyme a of and different with RNAP II in the RNAP to the and RNAP III to both the and we also to the in the associated proteins found in we found that complex is associated with of proteins that a in such diverse as elongation and ribosomal RNA We focused on the analysis of highly enriched complexes found in with RNA polymerase II, sensitivity inducing factor complex and transcription factor II data show that label-free quantitative proteomic analysis of protein complexes highly reproducible peptide level quantitation as well as in-depth information on associated proteins that provide a snapshot of RNA polymerase protein have that quantitation using label-free spectral counting is a highly reproducible at both the protein and peptide unique are differentially by the proteins have a higher of than other We have that this is for the of RNAPII with a higher of spectral unique protein on the of protein complex in the same such as the use of at known A.C. C. S. D. proteomic analysis of complexes using normalized spectral abundance 2006; PubMed Scopus Google Scholar). The use of other quantitative methods such as quantitation also not able to for these sampling proteins with are not Although the ability to proteins a biological is a in the level of spectral sampling reproducibility biological supports across quantitation by the same peptide in when complexes are under different cellular the of a peptide to using a proteomics in spectral sampling levels for when protein abundance We to for the in the level of spectral sampling by using the APEX protocol as previously reported S. C. R. R.D. R. The APEX protein quantitation from proteomics 2008; PubMed Scopus Google Scholar, P. C. R. X. protein the of and 2007; PubMed Scopus Google Scholar, C. and protein abundance from protein 2008; PubMed Scopus Google Scholar). In addition, we used different of data from the analysis of lysate and from replicate of a highly purified and related of protein complexes. of was also not able to the in spectral sampling observed for the different RNA polymerase by the that the peptide detectability models are using binary values not and for peptide detection in the data The use of discrete values as of spectral the model based on the we and that replicate analyses. in peptide detectability for the same protein shows the of using discrete values for computational used for peptide For this we that the for label-free studies is the of advanced computational that are able to the of spectral peptide both the of the and of protein abundance Toward this the data from our analysis is as in the methods addition, we have that the use of biological replicate analysis using quantitative label-free proteomics provide insight the of low level interacting proteins such as the of the protein complexes DSIF and TFIIF. of DSIF and TFIIF have been to with RNAPII the as most by A. M. M. Leike K. J. Cramer P. of the RNA polymerase II transcription PubMed Scopus Google Scholar). We have that the proteins these complexes higher in their values across biological when with replicates. The supports the that proteins that interact with a purified complex in a dynamic fashion in higher in the quantitation across different biological replicates. is by the that was with a of reproducibility across all present at levels than DSIF and TFIIF of quantitative methods to the abundance of proteins a we able to quantitation of low level associated proteins and complex We have that quantitation using label-free spectral counting is a highly reproducible at both the protein and peptide unique are differentially by the proteins have a higher of than other We have that this is for the of RNAPII with a higher of spectral unique protein on the of protein complex in the same such as the use of at known A.C. C. S. D. proteomic analysis of complexes using normalized spectral abundance 2006; PubMed Scopus Google Scholar). The use of other quantitative methods such as quantitation also not able to for these sampling proteins with are not Although the ability to proteins a biological is a in the level of spectral sampling reproducibility biological supports across quantitation by the same peptide in when complexes are under different cellular conditions. the of a peptide to using a proteomics in spectral sampling levels for when protein abundance We to for the in the level of spectral sampling by using the APEX protocol as previously reported S. C. R. R.D. R. The APEX protein quantitation from proteomics 2008; PubMed Scopus Google Scholar, P. C. R. X. protein the of and 2007; PubMed Scopus Google Scholar, C. and protein abundance from protein 2008; PubMed Scopus Google Scholar). In addition, we used different of data from the analysis of lysate and from replicate of a highly purified and related of protein complexes. of was also not able to the in spectral sampling observed for the different RNA polymerase by the that the peptide detectability models are using binary values not and for peptide detection in the data The use of discrete values as of spectral the model based on the we and that replicate analyses. in peptide detectability for the same protein shows the of using discrete values for computational used for peptide For this we that the for label-free studies is the of advanced computational that are able to the of spectral peptide both the of the and of protein abundance Toward this the data from our analysis is as in the methods In addition, we have that the use of biological replicate analysis using quantitative label-free proteomics provide insight the of low level interacting proteins such as the of the protein complexes DSIF and TFIIF. of DSIF and TFIIF have been to with RNAPII the as most by A. M. M. Leike K. J. Cramer P. of the RNA polymerase II transcription PubMed Scopus Google Scholar). We have that the proteins these complexes higher in their values across biological when with replicates. The supports the that proteins that interact with a purified complex in a dynamic fashion in higher in the quantitation across different biological replicates. is by the that was with a of reproducibility across all present at levels than DSIF and TFIIF of quantitative methods to the abundance of proteins a we able to quantitation of low level associated proteins and complex We to the of the and for and of this with with

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