Shao‐En Ong

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system. Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system. Proteomics, the large scale study of the protein complement of a cell or tissue, has its origins in the technology of two-dimensional (2D) 1The abbreviations used are: 2D, two-dimensional; ICAT, isotope-coded affinity tag; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tandem MS; 1D, one-dimensional. gel electrophoresis invented more than 25 years ago (1.O’Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar, 2.Klose J. Kobalz U. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome.Electrophoresis. 1995; 16: 1034-1059Google Scholar). In 2D gel electrophoresis, quantitation is achieved by recording differences in the staining pattern of proteins derived from two states of cell populations or tissues. Therefore, in addition to obtaining increasingly higher resolution, technology improvements in the 2D gel community have been directed toward the image analysis of 2D gels and the relative quantitation of protein spots by their intensity of staining (3.Gorg A. Obermaier C. Boguth G. Harder A. Scheibe B. Wildgruber R. Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients.Electrophoresis. 2000; 21: 1037-1053Google Scholar, 4.Herbert B.R. Harry J.L. Packer N.H. Gooley A.A. Pedersen S.K. Williams K.L. What place for polyacrylamide in proteomics?.Trends Biotechnol. 2001; 19: 3-9Abstract Full Text Full Text PDF Google Scholar, 5.Patton W.F. Beechem J.M. Rainbow’s end: the quest for multiplexed fluorescence quantitative analysis in proteomics.Curr. Opin. Chem. Biol. 2002; 6: 63-69Google Scholar, 6.Zhou G. Li H. DeCamp D. Chen S. Shu H. Gong Y. Flaig M. Gillespie J.W. Hu N. Taylor P.R. Emmert-Buck M.R. Liotta L.A. Petricoin III, E.F. Zhao Y. 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers.Mol. Cell. Proteomics. 2002; 1: 117-124Google Scholar). Mass spectrometry has long been used in a quantitative manner in the small molecule field (7.Browne T.R. Van Langenhove A. Costello C.E. Biemann K. Greenblatt D.J. Kinetic equivalence of stable-isotope-labeled and unlabeled phenytoin.Clin. Pharmacol. Ther. 1981; 29: 511-515Google Scholar). Pharmacological researchers, for example, use isotopically labeled analogs of the compound of interest and add a known amount to the sample for analysis. This is because mass spectrometry is not quantitative per se because of varying detector response, differential ionization yields for different substances, and other factors. Observed peak ratios for isotopic analogs, however, are highly accurate, because there are no chemical differences between the species, and they are analyzed in the same experiment. One of the first uses of isotopic labels in proteomics was for improved sequence assignment in peptide sequencing by tandem mass spectrometry by incorporating 18O atoms at the C terminus of a peptide (8.Shevchenko A. Chernushevich I. Standing K.G. Thompson B. Wilm M. Mann M. Rapid “de novo” peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer.Rapid Commun. Mass Spectrom. 1997; 11: 1015-1024Google Scholar, 9.Schnolzer M. Jedrzejewski P. Lehmann W.D. Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.Electrophoresis. 1996; 17: 945-953Google Scholar, 10.Uttenweiler-Joseph S. Neubauer G. Christoforidis S. Zerial M. Wilm M. Automated de novo sequencing of proteins using the differential scanning technique.Proteomics. 2001; 1: 668-682Google Scholar). The 18O technique had already been used in protein chemistry and was subsequently shown to have interesting uses in quantitation, as well (11.Mirgorodskaya O.A. Kozmin Y.P. Titov M.I. Korner R. Sonksen C.P. Roepstorff P. Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards.Rapid Commun. Mass Spectrom. 2000; 14: 1226-1232Google Scholar, 12.Yao X. Freas A. Ramirez J. Demirev P.A. Fenselau C. Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus.Anal. Chem. 2001; 73: 2836-2842Google Scholar, 13.Larsen M.R. Larsen P.M. Fey S.J. Roepstorff P. Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry.Electrophoresis. 2001; 22: 566-575Google Scholar, 14.Stewart I.I. Thomson T. Figeys D. 18O labeling: a tool for proteomics.Rapid Commun. Mass Spectrom. 2001; 15: 2456-2465Google Scholar). Structural biologists often employ 15N media, in which all 14N atoms are replaced by 15N, to determine phase shifts in NMR studies. Lahm and Langen (15.Lahm H.W. Langen H. Mass spectrometry: a tool for the identification of proteins separated by gels.Electrophoresis. 2000; 21: 2105-2114Google Scholar) and subsequently Chait and co-workers (16.Oda Y. Huang K. Cross F.R. Cowburn D. Chait B.T. Accurate quantitation of protein expression and site-specific phosphorylation.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6591-6596Google Scholar) used this 15N-substituted medium for the purpose of quantifying differences between states of microorganisms. The former group used MALDI and 2D gel electrophoresis to quantify the abundance of mixed spots in 2D gels of bacterial proteins, whereas the latter group quantified relative differences in phosphopeptide abundance in yeast. Although clearly showing the power of stable isotope labeling, the particular method employed was limited in its wider applications; 15N-substituted media are difficult and expensive to make for mammalian systems, so the method has generally been limited to microorganisms that can be grown in these media. Additionally, the degree of incorporation is not necessarily 100%, and because there are varying numbers of nitrogen atoms in the different amino acids, automated interpretation of the resulting spectra has proven difficult. Smith and co-workers (17.Veenstra T.D. Martinovic S. Anderson G.A. Pasa-Tolic L. Smith R.D. Proteome analysis using selective incorporation of isotopically labeled amino acids.J. Am. Soc. Mass Spectrom. 2000; 11: 78-82Google Scholar) have used fourier transform-ion cyclotron resonance (FTICR) measurements of intact proteins from microorganisms that were labeled with deuterated leucine-containing media. In this way the number of leucines could be estimated, which helped in the assignment of protein identity to a measured molecular weight (17.Veenstra T.D. Martinovic S. Anderson G.A. Pasa-Tolic L. Smith R.D. Proteome analysis using selective incorporation of isotopically labeled amino acids.J. Am. Soc. Mass Spectrom. 2000; 11: 78-82Google Scholar). In 1999 Aebersold and co-workers (18.Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Google Scholar) introduced the isotope-coded affinity tag (ICAT) method for relative quantitation of protein abundance. In this approach, an isotopically labeled affinity reagent is attached to particular amino acids in all proteins in the population. After digestion of the protein to peptides, as a necessary step in all mainstream proteomic protocols, the labeled peptides are affinity-purified using the newly incorporated affinity tag, thereby achieving a simplification of the peptide mixture at the same time as incorporating the isotopic label. The method has been applied to a range of problems such as the quantification of microsomal proteins in differentiated versus undifferentiated HL-60 cells (19.Han D.K. Eng J. Zhou H. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.Nat. Biotechnol. 2001; 19: 946-951Google Scholar). Limitations of the first iteration of the ICAT principle, which uses biotin as the affinity tag and cysteine as the reactive amino acid, include nonspecific binding to the streptavidin affinity matrix and multiple subsequent reactions at the same site. In recent improvements to the ICAT methodology the cysteines are reacted to solid beads, and a labeled amino acid is attached to the cysteine (20.Zhou H. Ranish J.A. Watts J.D. Aebersold R. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry.Nat. Biotechnol. 2002; 20: 512-515Google Scholar). This method addresses many of the above limitations and leads to a larger number of identifications of cysteine-containing peptides. However, the method is performed by cross-linking peptides to beads via their cysteine groups and photo-releasing them afterward, which may compromise low analysis. number of isotopic labeling have been that the of chemical of the peptides or proteins M. M. G. P. Quantitation and de novo sequencing of proteins by isotopic labeling of peptides with a Chem. 2000; Scholar, G. A. novo peptide sequencing and quantitative profiling of complex protein mixtures using abundance Biotechnol. 2002; 20: Scholar, A. Watts J.D. R. S. Eng P. Aebersold R. stable isotope labeling of peptides for quantitation and de novo sequence Commun. Mass Spectrom. 2001; 15: Scholar). of these the labeling and peptide step as in the ICAT method, whereas these two or not include the affinity step L. R. L. P. A. C. Thompson proteomics based on stable isotope labeling and affinity Mass Spectrom. 2002; Scholar). quantitation of proteins, labeling and affinity the directly have been Y. T. Chait B.T. analysis of proteins as a tool for the Biotechnol. 2001; 19: Scholar, H. Watts J.D. Aebersold R. approach to the analysis of protein Biotechnol. 2001; 19: Scholar, N.H. T.D. Smith R.D. isotope-coded affinity tag approach for and in Chem. 2001; 73: Scholar). In this we describe a stable isotope labeling that we SILAC isotope labeling by amino acids in cell essential amino acids are to amino acid cell culture media and are incorporated into all proteins as they are into the chemical labeling or affinity are and the method is with all cell culture We that incorporation is and that cells normal in the of labeled media. The method is and and is used in an example, we applied SILAC to the study of cells as they from into This process of muscle necessarily changes in the expression of proteins as the cells from cell to proteins were found to be up-regulated during this of these have not been as up-regulated proteins in this model of muscle differentiation. SILAC cells but may be other quantitative proteomics cell culture is The essential medium and in and was from number The medium was to the the medium was in with and the pH was to The amino acids and were as in and to the media to a of and The medium was a to medium in labeling or were as in and to the media for a of and cells were grown in essential medium supplemented with 2 and in a with in lines were grown for cell in labeling media either normal leucine or Leu-d3 the of differentiation. cells were grown to in normal leucine media. cells that were used for were grown in Leu-d3 media and were the of and the amount of in the media was to medium was replaced with medium every 2 a of were with to proteins and in a pH and The was for two of and to Protein quantitation was performed using the protein and mixtures of were in protein ratios of and the relative quantitation of protein expression during muscle of cell from different and were as After of protein with the mixtures of and samples were in the an undifferentiated at was mixed with an amount of protein from samples at and Protein mixtures were on a gel and to the gel were and to in-gel and digestion as A. Wilm M. Mann M. Mass spectrometric sequencing of proteins polyacrylamide Chem. 1996; Scholar, A. Mann M. of mass spectrometry to study 2000; Scholar). MALDI were with a and a with acid as the M. A. T. S. L. T. Mann M. sequencing of proteins from polyacrylamide gels by mass 1996; were and on a into J. R. Roepstorff P. and technique based on for the analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass Mass Spectrom. 1999; Scholar). were with in acid directly into a and the to and analysis on a tandem mass and with a Proteins were by peptide sequence tags M. Wilm identification of peptides in sequence by peptide sequence Chem. derived from spectra of peptides, the protein maintained and updated at the using the quantitative ratios in the and Leu-d3 isotope an isotopic was applied as after peptide the peptide sequence was to the tool which is of the The isotope pattern of the mass in the isotope was from the isotope pattern to the peak of the higher mass Mammalian cells a number of amino acids, these amino acids be in cell culture medium as amino acids for the medium to cell labeled analogs of these amino acids can be and are the labeled of an amino acid is of the abundance amino acid, be incorporated into newly protein After a number of cell of this particular amino acid have been replaced by its isotopically labeled there is no chemical between the labeled amino acid and the amino acid, the cells a control cell grown with the normal amino This is in The experimental cell can be in a specific such as or for Protein populations from samples are and because the is directly into the amino acid sequence of every the can be mixed proteins or peptides the of the labeled to unlabeled as no more is and no can place at the amino acid The proteins and peptides can be analyzed in any of the in which they are analyzed in Quantitation place at the of the peptide mass or peptide mass the same as in any other stable isotope method as is to that the of chemical the same and for SILAC as for SILAC with ICAT labeling, which is the well and method in quantitative proteomics by mass can be from the proteins to be and that can make difficult to the samples in directly states during multiple the chemical and affinity step can be difficult to with small of and peptides are to the in of a large number of affinity to be performed for a experiment. One between SILAC and ICAT methods is that SILAC labels more than of the peptides whereas ICAT labels more than This is based on the 2 and relative abundance of cysteine and and an of amino acids for peptides that can be by mass ICAT in of the peptide mixture whereas SILAC not the peptide resulting from a the in in the case of ICAT is based on the ability to cysteine is to proteins at in ICAT are by the functional group attached to the cysteine whereas in SILAC they are the same as for the unlabeled peptide We use of a labeling medium in amino acids, and for was to the normal amino acids with the of the which be labeled with We leucine in these because is the amino acid, between and and is essential amino acids could have been as mammalian cells media for their amino acids in the can be by the this we used of normal as not of amino the of we cells in media with Leu-d3 but supplemented with normal in place of the 2 clearly that proteins normal leucine can be the incorporation of Leu-d3 in proteins, accurate quantitation of labeled and unlabeled cells not be In the we we have used a essential medium supplemented with the essential amino acids and by SILAC with methods are to the of the amino acid used but are generally not a large of the of the experiment. The SILAC method not in cell culture the of media that we find generally to a of cell lines and that we have in example, we have grown other cell lines a cell cells cells and a cell in culture media not the of this method to any cell system. We performed a time to the time for cells to Leu-d3 in all proteins. The cells were grown in medium for different of shown in incorporation of Leu-d3 was in peptides after of larger incorporation of Leu-d3 was at time with incorporation by This to five doublings for used in this that cell lines can be for use in to protein be that in the time for the cells to five proteins with long incorporation of the as the cells protein to their We were to proteins by peptide mass as well as directed peptide sequencing with In mixtures of and samples were the identification of leucine-containing peptides was by the of peak in the mass the we were to these were and peak by the spectra and peptides to a sample a MS/MS, as in the from and peptides can to the identity of quantitation In peptide mass the of Leu-d3 in peptides peptides peptides a leucine have their peptide by and with more labeled have their mass by the was to and protein based on and peptides in However, the mixtures of proteins in a as well as the from the two cell the process of protein identification by peptide mass we the of mass spectrometric by mass spectra of and peptides were for the mass of fragments the leucine shifts in to the assignment of peptide sequence This is in to (8.Shevchenko A. Chernushevich I. Standing K.G. Thompson B. Wilm M. Mann M. Rapid “de novo” peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer.Rapid Commun. Mass Spectrom. 1997; 11: 1015-1024Google Scholar, 10.Uttenweiler-Joseph S. Neubauer G. Christoforidis S. Zerial M. Wilm M. Automated de novo sequencing of proteins using the differential scanning technique.Proteomics. 2001; 1: 668-682Google Scholar) incorporation of 18O in peptides to a that in obtaining sequence tag was performed using known of cell from were mixed in ratios of and The ratios of peak of different leucine-containing peptides were found to be in the proteins analyzed and of two peptides mixed in the in ratios are to We performed the not for the isotopic in peptides leucine the ratios were In we in higher differences than We this to be a of the of the peptide mixture in a gel and to be by that not an step of peptide by this this using nanoelectrospray, we to the relative of in the spectra from and Leu-d3 shown in the ratios from the relative from all the well with the of The ratios for a of are shown in I. is to that the in labeled and unlabeled peptides are and no are introduced because of the of a of peptides by their in a we could proteins in based on quantitative changes in their we used cells that have been used as an in model for muscle F. D. J.A. of is to in of protein kinase is for of expression and subsequent Biol. Chem. 1999; Scholar, S.J. A. of mammalian by 2000; Scholar, I. S. K. W. binding and of Biol. Chem. 2002; Scholar). Although the process of is not The of to can be by the cells from the cell expression of and of these cells to of J.A. expression an of and of muscle Biol. Scholar). of the changes that these cells as they in a medium low in changes in protein we the of cells in normal medium and the cells in medium to differentiate. were at different time and analyzed to determine the identity and in abundance of the proteins. the process of is by in cell morphology, and because of changes in expression of matrix proteins, and J.A. expression an of and of muscle Biol. Scholar, F. D.J. matrix is for muscle but not Cell. 1996; Scholar). the SILAC we differential protein expression could be measured in this system. we a combination of gel electrophoresis with mass from different time were separated by gel electrophoresis and or the gel of were in of the mixed which in with samples had shown differential staining between and other time of proteins were quantified in these was to ratios in different leucine-containing peptides for the same The process of quantitation was by the of the because of the large number of protein found in the protein mixture used for analysis. In such we to quantitation on peptide that were well separated and from for isotopic was applied necessary The protein quantitation are by in expression of was up-regulated on 2 and of muscle relative to example, glyceraldehyde-3-phosphate by The of of pyruvate kinase by which with the that the and are more highly in muscle than in other I. K. M. S. cells pyruvate kinase peptide can experimental in 2001; Scholar). Protein such as proteins were found to be up-regulated to in with protein during the of fibronectin, of the of matrix and essential for were found to be Although is known to be an essential in muscle cell F. D.J. matrix is for muscle but not Cell. 1996; Scholar, G. P. A. L. L. G. The is for by growth Biol. had not been shown to be up-regulated during this The relative of other proteins in such as were to the of as an internal have shown that the process of quantitation of protein by SILAC can be performed using standard and in proteomics and can be by groups with cell culture Although we have the of the method with gel electrophoresis and mass the higher in quantitative analysis and protein identification by are to the of this

Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments trypsin C-terminal to arginine lysine. non-tryptic the C-terminal of proteins of tryptic to mass accuracy to a of proteins identified with non-tryptic peptide peptide in be peptide identification be trypsin Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. 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