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Prevention of Amino Acid Conversion in SILAC Experiments with Embryonic Stem Cells

2008; Elsevier BV; Volume: 7; Issue: 9 Linguagem: Inglês

10.1074/mcp.m800113-mcp200

1535-9484

Sean C. Bendall, Chris Hughes, Morag Stewart, Bradley W. Doble, Mickie Bhatia, Gilles Lajoie,

CRISPR and Genetic Engineering

Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotope-coded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter l-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of "light" and "heavy" peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human. Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotope-coded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter l-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of "light" and "heavy" peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human. Large scale identification of proteins using mass spectrometry (MS)-based 1The abbreviations used are: MS, mass spectrometry; bFGF, basic fibroblast growth factor; DMEM, Dulbecco's Modified Eagle's Medium; hESC, human embryonic stem cell; FBS, fetal bovine serum; hESC, human embryonic stem cell; KOSR, knockout serum replacement; LC, liquid chromatography; MEF, mouse embryonic fibroblast; mESC, mouse embryonic stem cell; SILAC, stable isotope labeling with amino acids in culture; LC-MS, liquid chromatography mass spectrometry; LC-MS/MS, liquid chromatography tandem mass spectrometry. proteomics has become common place in recent years (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5602) Google Scholar). However, the generation of increasingly long lists of proteins now confronts researchers with the more challenging problem of determining the biological significance of particular proteins. For this reason, researchers are exploiting quantitative proteomic strategies to extract more meaningful information from their data sets. By comparing multiple samples and different conditions versus controls, it is possible to discern proteins that are altered in a given set of experimental or biological conditions from the thousands of proteins that are identified (2Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1318) Google Scholar). In MS-based proteomics, quantitative strategies typically involve the use of stable isotopic reagents to generate "heavy" and "light" samples, which retain their chemical identity and can then be differentiated and directly compared by MS analysis. Although chemical labeling methods such as ICAT (isotope-coded affinity tags) have demonstrated widespread applicability (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5602) Google Scholar), metabolic incorporation strategies such as stable isotope labeling with amino acids in cell culture (SILAC) are becoming more common for cell types that can be grown for extensive periods of time in vitro. Though there are numerous advantages for using SILAC-based methods, compared with chemical labeling, a major drawback is the unintended metabolic inter-conversion of isotopic amino acids in the labeling process, generating artifacts affecting the quantification. This is a particular problem with arginine, which is a metabolic precursor for proline biosynthesis (Fig. 1). In general, large scale SILAC experiments use both isotope-coded arginine and lysine to obtain labeling of all possible tryptic peptides thereby maximizing quantitative coverage of all potential peptides in a given experiment. Several studies using SILAC have described the problematic conversion of isotopically coded arginine to labeled proline in cells (3Hwang S.I. Lundgren D.H. Mayya V. Rezaul K. Cowan A.E. Eng J.K. Han D.K. Systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling.Mol. Cell Proteomics. 2006; 5: 1131-1145Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 4Ong S.E. Kratchmarova I. Mann M. Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC).J. Proteome Res. 2003; 2: 173-181Crossref PubMed Scopus (375) Google Scholar, 5Ong S.E. Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (686) Google Scholar, 6Schmidt F. Strozynski M. Salus S.S. Nilsen H. Thiede B. Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC).Rapid Commun. Mass Spectrom. 2007; 21: 3919-3926Crossref PubMed Scopus (28) Google Scholar, 7Van Hoof D. Pinkse M.W. Oostwaard D.W. Mummery C.L. Heck A.J. Krijgsveld J. An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics.Nat. Methods. 2007; 4: 677-678Crossref PubMed Scopus (106) Google Scholar) (Fig. 1), which results in an underestimation of the abundance of "heavy" tryptic peptides containing proline in a relative quantification experiment. This is of particular concern because ∼50% of all tryptic peptides between 700 and 6000 Da in the international protein index human data base contain at least one proline. Moreover, in a recent study it was reported that ∼30–40% of all observable proline containing peptides exhibited some level of conversion from heavy arginine (7Van Hoof D. Pinkse M.W. Oostwaard D.W. Mummery C.L. Heck A.J. Krijgsveld J. An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics.Nat. Methods. 2007; 4: 677-678Crossref PubMed Scopus (106) Google Scholar). Besides acting to skew the relative abundance observed, conversion of "heavy" arginine to "heavy" proline also further complicates the mass spectrometry data by increasing the number of peptide ion peaks. One strategy to minimize this problem is the reduction of the arginine concentration in the SILAC media rendering it metabolically unfavorable as a precursor for proline synthesis (8Blagoev B. Mann M. Quantitative proteomics to study mitogen-activated protein kinases.Methods. 2006; 40: 243-250Crossref PubMed Scopus (69) Google Scholar). Although this does reduce the conversion, it does not prevent it completely (3Hwang S.I. Lundgren D.H. Mayya V. Rezaul K. Cowan A.E. Eng J.K. Han D.K. Systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling.Mol. Cell Proteomics. 2006; 5: 1131-1145Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The other is to avoid the use of arginine altogether (3Hwang S.I. Lundgren D.H. Mayya V. Rezaul K. Cowan A.E. Eng J.K. Han D.K. Systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling.Mol. Cell Proteomics. 2006; 5: 1131-1145Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Finally, alternative corrections can be done mathematically, post-analysis, but these are inaccurate and do not solve the problem of increased peptide ion complexity (9Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar). Although reducing the concentration of available arginine in SILAC labeling media seems to be the method of choice for preventing its conversion to proline, this approach could be problematic for particular cells types like those with rapid cellular metabolism (10Scott L. Lamb J. Smith S. Wheatley D.N. Single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignant cells.Br. J. Cancer. 2000; 83: 800-810Crossref PubMed Scopus (146) Google Scholar). Moreover, certain sensitive cell types, such as human embryonic stem cells (hESCs) die or differentiate under restrictive metabolic conditions (7Van Hoof D. Pinkse M.W. Oostwaard D.W. Mummery C.L. Heck A.J. Krijgsveld J. An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics.Nat. Methods. 2007; 4: 677-678Crossref PubMed Scopus (106) Google Scholar, 11Hoffman L.M. Carpenter M.K. Characterization and culture of human embryonic stem cells.Nat. Biotechnol. 2005; 23: 699-708Crossref PubMed Scopus (382) Google Scholar). To this end, Van hoof et al. (7Van Hoof D. Pinkse M.W. Oostwaard D.W. Mummery C.L. Heck A.J. Krijgsveld J. An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics.Nat. Methods. 2007; 4: 677-678Crossref PubMed Scopus (106) Google Scholar) sought to compensate for arginine to proline conversion in SILAC experiments by advocating the use of isotopically labeled arginine (15N4-Arg) in light SILAC media as well as (15N4-13C6-Arg) in the heavy media. With the assumption that arginine to proline conversion would be the same under both conditions, the monoisotopic peak of light and heavy proline containing peptide ions could be compared with correctly determined relative abundance, as both would be reduced proportionately. This method does provide a solution but it further complicates the procedure by generating additional converted proline peptide ions in the MS spectra and by adding the cost of expensive isotopic reagents. Also, there is no guarantee that the rate of arginine to proline conversion will be the same when comparing different cell types or treatments. Adding to these difficulties is the fact that SILAC labeling of both hESCs (7Van Hoof D. Pinkse M.W. Oostwaard D.W. Mummery C.L. Heck A.J. Krijgsveld J. An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics.Nat. Methods. 2007; 4: 677-678Crossref PubMed Scopus (106) Google Scholar) and ESCs from the mouse (mESC) (12Graumann J. Hubner N.C. Kim J.B. Ko K. Moser M. Kumar C. Cox J. Schoeler H. Mann M. SILAC-labeling and proteome quantitation of mouse embryonic stem cells to a depth of 5111 proteins.Mol. Cell Proteomics. 2007; 7: 672-683Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) requires the use of mouse embryonic fibroblast (MEF) feeder cells, which precludes the attainment of high cell numbers necessary for large scale, SILAC-based proteomic applications (8Blagoev B. Mann M. Quantitative proteomics to study mitogen-activated protein kinases.Methods. 2006; 40: 243-250Crossref PubMed Scopus (69) Google Scholar, 13Andersen J.S. Lam Y.W. Leung A.K. Ong S.E. Lyon C.E. Lamond A.I. Mann M. Nucleolar proteome dynamics.Nature. 2005; 433: 77-83Crossref PubMed Scopus (948) Google Scholar, 14Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (367) Google Scholar, 15Selbach M. Mann M. Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK).Nat. Methods. 2006; 3: 981-983Crossref PubMed Scopus (211) Google Scholar, 16Yocum A.K. Gratsch T.E. Leff N. Strahler J.R. Hunter C.L. Walker A.K. Michailidis G. Omenn G.S. O'Shea K.S. Andrews P.C. Coupled global and targeted proteomics of human embryonic stem cells during induced differentiation.Mol. Cell Proteomics. 2008; 7: 750-767Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Faced with these challenges, we sought a simple solution, for arginine to proline conversion in SILAC experiments that would be compatible with a robust hESC culture protocol free of feeder cells. In this study, we demonstrate that by supplementing SILAC media with standard l-proline, the conversion of isotope-coded arginine to labeled proline can be prevented. In addition, when providing sufficient amounts of arginine, no back conversion from the increased proline supplement was observed. When proline supplementation of SILAC media was adapted to both mouse and human ESC culture reagents complete labeling with isotope-coded lysine and arginine can be routinely achieved using standard procedures in the absence of feeder cells without compromising the ESC phenotype. All SILAC media formulations and labeling procedures were modified from previously described protocols (5Ong S.E. Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (686) Google Scholar, 17Ong 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 (4584) Google Scholar). For all SILAC labeling experiments embryonic stem cell qualified Dulbecco's Modified Eagle's Medium (DMEM; 4500 mg/liter glucose, with sodium pyruvate, and no l-glutamine) was custom ordered without lysine or arginine (Specialty Media, Millipore). Before use, media was re-supplemented with 2.5 mm l-glutamine (Invitrogen), 0.798 mm isotope-coded l-lysine (13C6, 15N2), and 0.398 mm isotope-coded l-arginine (either 13C6 or 13C6, 15N4). In the case of light media, standard l-lysine and l-arginine were used. For HeLa cell culture the DMEM was supplemented with 10% (v/v) dialyzed fetal bovine serum (FBS), 1% (v/v) penicillin-streptomycin (both Invitrogen), and 0–800 mg/liter l-proline (Sigma). The DMEM for ESC cell culture was supplemented with 0.1 mm 2-mercaptoethanol, 1% non-essential amino acids (Invitrogen), and 20% or 15% Knockout Serum Replacement (KOSR; Invitrogen) reagent for hESC or mESC culture, respectively. Note that ESC media with 1% non-essential amino acids will have 11.5 mg/liter proline whereas KOSR at 20 and 15% (v/v) will contribute an additional 800 and 600 mg/liter l-proline, respectively (18Price, P. J., Goldsborough, M. D., and Tilkin, M. L. (1998) Embryonic stem cell serum replacement. International patent Application WO98/30679 p. 28Google Scholar). All media were filter-sterilized at 0.2 μm following formulation. To monitor isotopic amino acid incorporation and conversion, trypsin digests were prepared from whole cell lysates for LC-MS analysis. Cell culture media was removed, and cells were washed with phosphate-buffered saline. Cells were then mechanically removed from the plate, lysed by dissociation in 8 m urea, and 50 mm ammonium bicarbonate and frozen at −80 °C. The protein content was determined by Bradford assay, and where indicated, light and heavy isotopic samples were mixed (1:1) based on total protein concentration prior to digestion. For trypsin digestion, samples were reduced with 10 mm dithiothreitol, alkylated with 30 mm iodoacetamide, diluted 1:4 with 50 mm NH4CO3, and treated with modified trypsin (1:25 enzyme/substrate ratio; Promega) at 37 °C overnight. The resulting tryptic peptides were extracted using a 1-ml C18 solid phase extraction cartridge (Waters), eluted with 50% (v/v) acetonitrile, 0.1% (v/v) formic acid to remove urea and salts and were re-concentrated in a vacuum centrifuge. Dried fractions were reconstituted in 10% formic acid for LC-MS/MS analysis. Between 1.0 and 0.5 μg of each original sample was injected on a NanoAcquity UPLC (ultra performance liquid chromatography) (Waters) equipped with a 15 cm × 75 μm C18 reverse phase column employing a 90-min LC gradient (5–40% ACN, 0.1% formic acid) and detected in a data-dependent acquisition mode by tandem MS (Q-ToF Ultima; Waters). The MS was directed to use the following data-dependent acquisition parameters: survey scans range 400–1800 m/z, 1 s scans, 1–4 precursors selected based on charge state (+2, +3 and, +4 ions). MS/MS fragmentation was then performed on the ions using the charge-state collision energy profile function. Raw data files were processed using Protein Lynx Global Server 2.2.5 and summarized to peak list files (pkl). The processed data was searched against the human or mouse international protein index data base version 3.40 (appended with common protein contaminants including human keratin, bovine albumin, and trypsin) using the built in Protein Lynx Global Server search engine. The search parameters were: 100 ppm for MS and 0.1 Da for MS/MS, 2 missed cleavages on tryptic peptides, carbamidomethyl Cys as a fixed modification, and oxidized Met as a variable modification. For isotopic labeling, variable modifications of 5 or 6 Da for proline, 6 or 10 Da for arginine, and 8 Da for lysine were included in the search where appropriate. Quantification of peak areas was performed manually using MassLynx software version 4.1 on peptides indicated. MS spectra containing peptide ions of interest were summed, and background was subtracted using a polynomial order of 5 at 10% below the curve. The peak area was determined by integration using the built-in algorithm in MassLynx 4.1 to output peak area values. Peak area of the monoisotopic ions was used for comparison and determination of intensity ratios of peptide candidates. HeLa cells were passaged twice at a ratio of 1:8 in SILAC media containing the indicated amount of l-proline to ensure maximum incorporation of the isotopic arginine label (either 13C6 or 13C6, 15N4-Arg). SILAC media was changed every 3 days or as needed based on phenol red indicator. Whole cell lysates were digested and subject to LC-MS/MS in the initial sample analysis. Both potential and confirmed (by MS/MS) proline containing peptides resulting from the conversion of isotopic arginine were noted. All samples were then re-run in triplicate using the "include only" function to target previously detected/suspected proline containing peptides across all the samples. The candidate list of proline containing peptides was then used to follow the conversion of arginine to proline over varying proline supplement concentrations. Peptides containing labeled proline derived from isotope-coded arginine were confirmed by MS/MS in the 0 and 50 mg/liter proline samples. 13 proline-converted peptides observed in the replicate analysis and confirmed by MS/MS were used to monitor isotopic arginine to proline conversion in these experiments. These were: VAPEEHPVLLTEAPINPK, LILPGELAK, PMFIVNTNVPR, NTGIICTIGPASR, MSVQPTVSLGGFEITPPVVLR, PPYTVVYFPVRGR, YSSLVPIEK, AVFPSLVGRPR, VAPEEHPVLLTEAPINPK, SSGPYGGGGQYFAKPR, QYPKVPR, LAVNMVPFPR, and SSRAGLQFPVGR. To investigate the possible back conversion of proline to arginine, a similar process was repeated except the arginine containing peptide candidates did not have a proline in their sequence, so that proline conversion could not interfere with arginine incorporation calculations. Based on replicate analysis, those peptides used for arginine incorporation that were consistently observed and confirmed MS/MS were: STELLIR, AGTGVDNVDLEAATR, VIGSGCNLDSAR, TIAQDYGVLKADEGISFR, GKVKVGVDGFGR, and GITINAAHVEYSTAAR. For SILAC labeling of both hESC and mESC cultures measures were taken to remove all non-isotopic Arg and Lys from the system. All cells were washed with either phosphate-buffered saline or DMEM lacking Arg and Lys prior to exposure to SILAC media. Any reagents requiring dilution with DMEM were prepared in DMEM lacking Arg and Lys as well. hESC lines H1 and H9 (19Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12376) Google Scholar) were maintained in feeder cell-free culture on 1:15 Matrigel-coated plates (BD Biosciences). hESC SILAC media was supplemented with 4 ng/ml basic fibroblast growth factor (bFGF; Invitrogen) and pre-conditioned on irradiated mouse embryonic fibroblasts (MEFs) according to previously established protocols (20Xu C. Inokuma M.S. Denham J. Golds K. Kundu P. Gold J.D. Carpenter M.K. Feeder-free growth of undifferentiated human embryonic stem cells.Nat. Biotechnol. 2001; 19: 971-974Crossref PubMed Scopus (1599) Google Scholar). The MEF conditioned hESC SILAC media was further supplemented with 8 ng/ml bFGF prior to addition to hESC cultures and changed daily. Cells were passaged every 5–7 days through dissociation with 200 units/ml collagenase IV (Invitrogen). hESCs were cultured for two passages (>12 days) in SILAC media to achieve maximum isotopic amino acid incorporation. Analysis of hESC culture integrity and expression of pluripotent cell surface markers was performed as described previously (21Bendall S.C. Stewart M.H. Menendez P. George D. Vijayaragavan K. Werbowetski-Ogilvie T. Ramos-Mejia V. Rouleau A. Yang J. Bosse M. Lajoie G. Bhatia M. IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro.Nature. 2007; 448: 1015-1021Crossref PubMed Scopus (507) Google Scholar). E14K mESC cultures from 129/Ola mice were also maintained in the absence of feeder cells on 0.1% (v/v) gelatin-coated plates (Sigma) as described previously (22Doble B.W. Patel S. Wood G.A. Kockeritz L.K. Woodgett J.R. Functional redundancy of GSK-3alpha and GSK-3beta in Wnt/beta-catenin signaling shown by using an allelic series of embryonic stem cell lines.Dev. Cell. 2007; 12: 957-971Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). mESC SILAC media was supplemented with 1000 units/ml leukemia inhibitory factor (ESGROW, Millipore) prior to addition to cultures. Cells were passaged 1:3 every 2 days through dissociation in 0.25% trypsin, 2.21 mm EDTA (Wisent). mESCs were cultured for 4 passages (>8 days) in SILAC media to achieve maximum isotopic amino acid incorporation. To monitor isotope-coded amino acid incorporation and determine the optimum times for SILAC adaptation a list of both Lys and Arg containing candidate peptides for both human and mouse ESCs was generated. Those candidate peptides that were consistently identified by MS/MS across all samples during the labeling time course were: NLPQYVSNELLEEAFSVFGQVER, IHFPLATYAPVISAEK, SLQDIIAILGMDELSEEDKLTVSR, LISWYDNEFGYSNR, GVDEVTIVNILTNR, SSGPTSLFAVTVAPPGAR, ALMLQGVDLLADAVAVTMGPK, EVAAFAQFGSDLDAATQQLLSR, LLEDGEDFNLGDALDSSNSMQTIQK, EKPYFPIPEEYTFIQNVPLEDR, VGKDELFALEQSCAQVVLQAANER, GFGFVTYATVEEVDAAMNARPHK, VVLAYEPVWAIGTGK, and YPIEHGIITNWDDMEK for hESC; and KATGPPVSELITK, ALLFVPR, GLFIIDDK, YQAVTATLEEK, ALMLQGVDLLADAVAVTMGPK, SGETEDTFIADLVVGLCTGQIK, MVNHFIAEFK, IGYPAPNFK, IAIYELLFK, ALYETELADARR, SLMDEVVKATSR, ERPPNPIEFLASYLLK, TKVHAELADVLTEAVVDSILAIR for mESCs. In mammalian cells, the metabolism of arginine and proline are related, and each can serve as precursors for one another through slightly different but related pathways (Fig. 1c; reviewed in (23Morris Jr., S.M. Enzymes of arginine metabolism.J. Nutr. 2004; 134 (discussion 2765S–2767S): 2743S-2747SCrossref PubMed Google Scholar)). In a related pathway, both glutamate and glutamine can be precursors for arginine biosynthesis and products of arginine metabolism (Fig. 1c). However, only artifacts of proline synthesis from isotopic arginine have been reported in SILAC experiments. This observation raised the question of availability of amino acids in vitro cell culture media, particularly in SILAC formulations because previous in vivo mammalian studies have demonstrated that the inter-conversion of amino acids in this pathway (Fig. 1c) is primarily dependent on their bioavailability (24Urschel K.L. Rafii M. Pencharz P.B. Ball R.O. A multitracer stable isotope quantification of the effects of arginine intake on whole body arginine metabolism in neonatal piglets.Am. J. Physiol. Endocrinol. Metab. 2007; 293: E811-E818Crossref PubMed Scopus (35) Google Scholar). Because l-proline is a non-essential amino acid, its concentration in the two main basal media used in standard SILAC experiments, RPMI and DMEM, is only 20 and 0 mg/L, respectively. At the same time, the typical concentration of arginine, a metabolic precursor of proline (Fig. 1b), is 200 and 84 mg/liter, respectively. Based on this we surmised that the arginine to proline conversion artifacts in SILAC experiments were a direct result of the low or absent concentration of proline in standard SILAC labeling formulations. In standard DMEM-based SILAC media, in the absence of proline supplement, cells readily convert isotopic arginine to proline, which is then incorporated into newly synthesized proteins. This conversion divides the MS signal of the heavy proline containing peptide in a complex fashion, resulting in false relative abundance values when compared with an equal amount of light sample (Fig. 1, a and b). To eliminate this artifact in arginine-based SILAC procedures we investigated whether increased l-proline supplementation could prevent the conversion in a concentration-dependent manner. Equal amounts of cell lysates extracted from HeLa cells cultured in SILAC media (13C6-Arg) containing 0–800 mg/liter proline were digested with trypsin and subjected to LC-MS/MS analysis. Detected proline conversion peptides were used to build a list of peptide candidates for quantification. By using +2 or +3 charge states, the selection of the peptide candidates was based on whether they were observed in all samples, the number of proline residues, the extent of conversion observed, and whether the sequence contained arginine. Based on the triplicate analysis of all lysates, a list of thirteen peptide candidates was chosen and quantified across the 0–800 mg/liter proline concentrations. Throughout the titration of l-proline in SILAC media both signals of the converted proline containing peptides was monitored (Fig. 2, a and b). In addition, the ratio of heavy labeled Arg peptide to light peptide was measured to detect whether a decrease in the converted proline peptide signal resulted in a subsequent increase in the expected heavy peptide (Fig. 2, a and b). Within the set of peptide candidates proline conversion consumed an average of 28% of the monoisotopic signal of the heavy arginine peptide in the 0 mg/liter proline sample (standard SILAC media) (5Ong S.E. Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (686) Google Scholar). As the proline concentration was increased to 50, 100, and 200 mg/liter, the monoisotopic heavy proline peak occupied 9, 3, and 2%, respectively. However, within this list of proline-containing peptides no peaks corresponding to a converted proline could be detected in the MS spectrum beyond 100 mg/liter proline. As such, 2% of the total signal seen at 200 mg/liter proline was a result of unsubtracted background in the MS spectrum where there was low signal-to-noise. Moreover, maximum relative signal of heavy arginine-labeled peptide (∼20% increase versus 0 mg/liter Pro) was seen at 200 mg/liter and beyond (Fig. 2b). Consequently, even in some of the worst cases of proline conversion (Fig. 2c) no proline conversion could be observed in cells supplemented with 200 mg/liter proline or greater (d). Taken toge