A Protein Epitope Signature Tag (PrEST) Library Allows SILAC-based Absolute Quantification and Multiplexed Determination of Protein Copy Numbers in Cell Lines

Mass spectrometry-based proteomics increasingly relies on relative or absolute quantification. In relative quantification, stable isotope based methods often allow mixing at early stages of sample preparation, whereas for absolute quantification this has generally required recombinant expression of full length, labeled protein standards. Here we make use of a very large library of Protein Epitope Signature Tags (PrESTs) that has been developed in the course of the Human Protein Atlas Project. These PrESTs are expressed recombinantly in E. coli and they consist of a short and unique region of the protein of interest as well as purification and solubility tags. We first quantify a highly purified, stable isotope labeling of amino acids in cell culture (SILAC)-labeled version of the solubility tag and use it determine the precise amount of each PrEST by its SILAC ratios. The PrESTs are then spiked into cell lysates and the SILAC ratios of PrEST peptides to peptides from endogenous target proteins yield their cellular quantities. The procedure can readily be multiplexed, as we demonstrate by simultaneously determining the copy number of 40 proteins in HeLa cells. Among the proteins analyzed, the cytoskeletal protein vimentin was found to be most abundant with 20 million copies per cell, while the transcription factor and oncogene FOS only had 6000 copies. Direct quantification of the absolute amount of single proteins is possible via a SILAC experiment in which labeled cell lysate is mixed both with the heavy labeled solubility tag and with the corresponding PrEST. The SILAC-PrEST combination allows accurate and streamlined quantification of the absolute or relative amount of proteins of interest in a wide variety of applications. Mass spectrometry-based proteomics increasingly relies on relative or absolute quantification. In relative quantification, stable isotope based methods often allow mixing at early stages of sample preparation, whereas for absolute quantification this has generally required recombinant expression of full length, labeled protein standards. Here we make use of a very large library of Protein Epitope Signature Tags (PrESTs) that has been developed in the course of the Human Protein Atlas Project. These PrESTs are expressed recombinantly in E. coli and they consist of a short and unique region of the protein of interest as well as purification and solubility tags. We first quantify a highly purified, stable isotope labeling of amino acids in cell culture (SILAC)-labeled version of the solubility tag and use it determine the precise amount of each PrEST by its SILAC ratios. The PrESTs are then spiked into cell lysates and the SILAC ratios of PrEST peptides to peptides from endogenous target proteins yield their cellular quantities. The procedure can readily be multiplexed, as we demonstrate by simultaneously determining the copy number of 40 proteins in HeLa cells. Among the proteins analyzed, the cytoskeletal protein vimentin was found to be most abundant with 20 million copies per cell, while the transcription factor and oncogene FOS only had 6000 copies. Direct quantification of the absolute amount of single proteins is possible via a SILAC experiment in which labeled cell lysate is mixed both with the heavy labeled solubility tag and with the corresponding PrEST. The SILAC-PrEST combination allows accurate and streamlined quantification of the absolute or relative amount of proteins of interest in a wide variety of applications. MS-based proteomics has become a method of choice to study proteins in a global manner (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5585) Google Scholar, 2Cravatt B.F. Simon G.M. Yates 3rd, J.R. The biological impact of mass-spectrometry-based proteomics.Nature. 2007; 450: 991-1000Crossref PubMed Scopus (571) Google Scholar, 3Gstaiger M. Aebersold R. Applying mass spectrometry-based proteomics to genetics, genomics and network biology.Nat. Rev. Genet. 2009; 10: 617-627Crossref PubMed Scopus (334) Google Scholar). Mass spectrometry is not inherently quantitative but many methods have been developed to overcome this limitation. Most of them are based on stable isotopes and introduce a mass shifted version of the peptides of interest, which are then quantified by their “heavy” to “light” ratio. Stable isotope labeling is either accomplished by chemical addition of labeled reagents, enzymatic isotope labeling, or metabolic labeling (4Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1317) Google Scholar, 5Bachi A. Bonaldi T. Quantitative proteomics as a new piece of the systems biology puzzle.J. 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For example, stable isotope labeling by amino acids in cell culture (SILAC) 1The abbreviations used are:SILACstable isotope labeling of amino acids in cell cultureAQUAabsolute quantificationPSAQprotein standard absolute quantificationQconCATquantification concatamerPrESTprotein epitope signature tagAIFall ion fragmentationABPalbumin binding protein. 1The abbreviations used are:SILACstable isotope labeling of amino acids in cell cultureAQUAabsolute quantificationPSAQprotein standard absolute quantificationQconCATquantification concatamerPrESTprotein epitope signature tagAIFall ion fragmentationABPalbumin binding protein. (7Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar, 8Mann M. Functional and quantitative proteomics using SILAC.Nat. Rev. Mol. Cell Biol. 2006; 7: 952-958Crossref PubMed Scopus (759) Google Scholar) is performed by metabolic incorporation of light or heavy labeled amino acids into the proteome. Labeled proteomes can also be used as internal standards for determining protein levels of a cell or tissue proteome of interest, such as in the spike-in SILAC approach (9Geiger T. Wisniewski J.R. Cox J. Zanivan S. Kruger M. Ishihama Y. Mann M. Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics.Nat. Protoc. 2011; 6: 147-157Crossref PubMed Scopus (225) Google Scholar). stable isotope labeling of amino acids in cell culture absolute quantification protein standard absolute quantification quantification concatamer protein epitope signature tag all ion fragmentation albumin binding protein. stable isotope labeling of amino acids in cell culture absolute quantification protein standard absolute quantification quantification concatamer protein epitope signature tag all ion fragmentation albumin binding protein. Absolute quantification is technically more challenging than relative quantification and can only be performed accurately for a single or a small number of proteins at a time (10Brun V. Masselon C. Garin J. Dupuis A. Isotope dilution strategies for absolute quantitative proteomics.J. Proteomics. 2009; 72: 740-749Crossref PubMed Scopus (262) Google Scholar). 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Biol. 2005; 6: PubMed Scopus Google Scholar) are with heavy isotopes and spiked in sample peptides are but when many peptides or proteins to be the from quantification that are of spiking in of the standard sample and enzymatic which is a in the Furthermore, any of the quantification The QconCAT approach is based on artificial proteins that are of artificial protein is recombinantly expressed in coli and spiked into the sample QconCAT in allows of labeled peptides but not for protein or in the proteins the The absolute SILAC and FlexiQuant approaches these by labeling full length proteins by heavy of the amino acids and and FlexiQuant in proteins in or in cell whereas absolute SILAC was with recombinant protein expression in E. The protein standard is at early such as to cell sample can be performed in and the SILAC protein is with the proteome these at the of to full length proteins, which and generally these methods to proteins. In this study we the absolute SILAC approach by use of a highly and for protein standard We short Protein Epitope Signature Tags which are in a manner by the Human Protein Atlas and used as for M. E. C. B. E. A. C. H. D. M. A. C. R. J. M. A. I. C. M. M. G. C. M. J. J. E. J. I. E. J. A. R. J. M. A. A. Steen J. M. F. S. M. H. S. E. A. J. H. J. U. S. F. protein for and based on proteomics.Mol. Cell. Proteomics. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. C. A. C. J. E. S. H. F. M. a proteome of for tissue 2005; PubMed Scopus Google Scholar, E. A. A. J. H. E. A. S. C. S. F. M. Human Protein Atlas for expression based on Cell. Proteomics. 2008; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). The of the Human Protein Atlas is to at to all proteins and to use this to study the tissue and the of the proteome F. M. E. A. E. S. C. S. J. A. H. M. global of protein expression in and Biol. 2009; PubMed Scopus Google Scholar, E. 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The PrESTs and mixed and in in with and with The protein was with for with and with The was by with and on J. Ishihama Y. Mann M. and for and sample in Chem. 2003; PubMed Scopus Google Scholar). of the PrESTs spiked into HeLa was performed on mass to via a ion The peptides on a 20 with and with a from The mass was in a to and full or to with a of at The most abundant and using by in the J. E. E. O. D. M. M. A. Mann M. S. ion with very Cell. Proteomics. 2009; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). The ratios of the light PrEST heavy peptides on the with using the as The peptides with a with over 40 The mass peptides with all ion fragmentation by of the and all ion fragmentation using fragmentation T. Cox J. Mann M. on mass using Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). at a of at The heavy PrESTs light peptides using the a of to a The in of and a of was to the peptides into the mass sample is to a single on the standard fragmentation method was for full for quantification A. R. G. M. Cox J. Bonaldi T. Mann M. allow protein on mass J. Mass 2007; Scopus Google Scholar). with J. Mann M. identification mass and protein 2008; PubMed Scopus Google Scholar) using the and the of the solubility tag to this For identification we used a in to the J. A. Mann M. a into the Proteome Res. 2011; 10: PubMed Scopus Google Scholar). of was in the as a and as well as as We and required a of amino acids per The mass for or was to and to by to For of the the and The was by a and was to for peptides by the between in which in a time of the based on the accurate The was as that to per with a of The ion mass was with the possible ion on the of the of at T. Cox J. Mann M. on mass using Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). Absolute amount of and was by The from and performed to the The HeLa in or with The by and using the For the the was by a and to concentration by a standard relative quantification, absolute quantification is a that of the absolute amount of the standard and the relative amount of the standard with the of and of the of standard is by and can be the that the of the we first a method to determine the absolute amount of each PrEST with we a of PrESTs and the of the SILAC PrEST method to accurately quantify cellular proteins. We then the to determine the copy numbers of 40 proteins in HeLa a cell we for the quantification of single proteins of interest, in which the are into one PrEST is to the a tag of amino In of in 40 peptides with a length between and amino acids We recombinantly expressed a heavy SILAC labeled version of the protein we used a approach based on and a tag to highly protein and to that only full length was The absolute concentration of protein was by amino which is the most accurate method for protein quantification, but which is only to highly proteins in large SILAC incorporation into was and its was as by mass spectrometry these in a and of the small of the the concentration of was not for of many readily peptides of the PrESTs from the Protein Atlas was mixed with a amount of labeled as in to allow for a SILAC this experiment a for each PrEST it was to be for the We to this on and than on a The we the peptides by T. Cox J. Mann M. on mass using Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) in at labeled peptides be quantified the corresponding peptides from the to a of of for PrEST quantification overcome the of the PrESTs which the heavy PrESTs by on of these simple mixtures of peptides using sample The a of over the based of a PrEST quantification can be at this a is this was not the of PrEST quantification was to be than that of the in the of from the PrEST quantification are in The of the SILAC ratios is used for the of such as the in the PrESTs for proteins and which had very to that the quantification not on the cellular or any of the target the amounts of PrEST is used in each PrEST quantification quantification in also not on the of the PrEST method the concentration of PrEST and not of protein. quantified the PrEST amounts we to protein expression levels in HeLa cells. For we first used PrESTs and quantified heavy SILAC labeled HeLa cells. cell lysates consist of of of the addition of a single or a large number of PrESTs not the of the the of the quantitative amounts we mixed PrESTs In we used mixtures of which spiked into HeLa lysate in The SILAC ratios levels of each PrEST in the such that the SILAC ratios the most accurately to one to The with levels of all the PrESTs was spiked into the lysate of SILAC labeled cells. The was to the by and in that with on mass We to quantify 40 of the proteins by PrEST generally quantified with PrEST peptides and to of example, the protein was quantified with which each quantification of the quantified peptides of had very SILAC one had a that by from the is and its to the it from to that we protein quantification on the of the the to the protein expression and the the in this For the of the endogenous proteins in the region by the PrEST which to the protein the of this of absolute protein quantification, we the ratios from internal or to from the of the one or These peptides are very for standard based methods such as but in very ratios for such that for PrEST and endogenous protein from in the SILAC-PrEST approach these peptides can quantification of peptides and peptides with with one or as well as their ratios are The ratios of the on by which is in the of of peptides from one in a new the of with both of the absolute quantification we the more including PrEST quantification and as well as of cellular of the target proteins. that the standard of the with all are on is and to the most accurate determination of cellular expression levels more the of each of the in the for each of the proteins are from the all protein expression can be and or to the of of protein expression determination from the the to the of the protein that the of the the whereas of SILAC determination of the to in the copy numbers per HeLa The the protein copy numbers per cell in The to the with copy numbers from 6000 to per cell quantified also we used the absolute for protein amounts in HeLa cell lysate to the corresponding copy numbers in cells. HeLa numbers in a cell the amount of each PrEST and their SILAC ratios with to the endogenous proteins we the cellular copy numbers of 40 proteins. of absolute quantification to a standard of was for of 40 proteins copy numbers per HeLa of the for the in protein protein protein protein protein protein body protein protein complex complex protein factor complex protein protein protein protein protein complex protein protein of the for the in in a new copy numbers are only for very proteins and it is to these copy numbers to the of the proteins The cytoskeletal protein vimentin and was the most abundant protein with 20 million copies per the the transcription factor and oncogene FOS is in 6000 copies in HeLa cell sample. proteins in cell are generally expressed at the factor protein protein is at only copies. with a as very highly expressed million of the have copies in this cell and whereas a has that not all protein have The only it is that proteins in their are in such copy of the has million copies per HeLa than a of the The is a of a and as it has a very copy number a is expressed at million whereas is expressed expression numbers be for metabolic These are but they that of the absolute expression levels of cellular proteins can to the of their in the we used expressed and PrESTs and quantified heavy protein and heavy cell to determine copy numbers in cell in applications it be more to heavy labeled which can then be mixed into any proteome of tissue and body samples. absolute quantification approach to we expressed of the PrESTs in heavy SILAC labeled E. them and a heavy quantification of PrEST we developed spiking the heavy into labeled quantification of the proteins, with to the experiment We also to a on the SILAC-PrEST to quantify single protein In this the in absolute protein quantification can be into one as in amount of the solubility tag is mixed into cell lysate with the labeled PrEST. of the sample then SILAC ratios of light solubility tag to labeled PrEST These ratios accurately quantify the amount of PrEST that was The also the ratios of labeled PrEST peptides to the endogenous protein these ratios quantify the absolute amount of endogenous protein in a single experiment that labeling is not required in this approach the ratios are of the PrEST the solubility tag region the and the protein PrEST region the endogenous method for quantification was performed for HeLa proteins in which the cell lysate and was quantified in in these based on The absolute levels generally well with the copy numbers in the experiment between the of both is a standard method in to determine absolute or at to determine protein We the SILAC-PrEST method to this the for cell lysate and as by the than of the amount quantified by that is by these peptides are The of the to in not the The solubility was by the which was to most of the sample a concentration of protein amount the absolute amounts with mass spectrometry the by the use of We also the levels of the transcription factor and FOS by the protein quantified in Here not to be and we between quantitative by and by using Here we have developed methods to determine the absolute levels of proteins in by of the absolute SILAC as well as the of a large library of PrEST protein PrESTs solubility which recombinant expression of PrESTs a wide variety of cellular We found that this solubility tag is it a large number of readily peptides a highly and accurately quantified of the tag it can be used to quantify all PrESTs in the of the PrEST is not a quantification is only performed the PrEST and not possible E. coli or PrESTs have been of the proteome and for their is in all PrESTs have been for target proteins, they in be for any in the Furthermore, in many more than one PrEST has been for the protein to allow for the of and of M. E. M. M. C. S. H. F. a Human Protein PubMed Scopus Google Scholar). PrESTs be in the are not for MS-based quantification. For of a SILAC-PrEST we the amount of PrESTs in a In the it be to the amount of protein from the from proteome PrESTs spiked in at corresponding amounts be in the in most The of the SILAC-PrEST approach can be for each in the procedure and it to be by is that of all in the procedure can be in the Here we have applications of for copy number determination in cell the be in the to quantify proteins from any and we to this We for with for for and for The E. coli for and was a from of The was to as absolute and is via with