Nic1 Inactivation Enables Stable Isotope Labeling with 13C615N4-Arginine in Schizosaccharomyces pombe

Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, 13C615N4-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of 13C615N4-arginine is catabolized by arginase and urease activity to 15N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni2+-dependent urease activity, through deletion of the sole Ni2+ transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable 13C615N4-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe. Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, 13C615N4-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of 13C615N4-arginine is catabolized by arginase and urease activity to 15N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni2+-dependent urease activity, through deletion of the sole Ni2+ transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable 13C615N4-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) 1The abbreviations used are:SILACstable isotope labeling by amino acids in cell cultureYESsolid yeast extract agarEMMEdinburgh minimal media. 1The abbreviations used are:SILACstable isotope labeling by amino acids in cell cultureYESsolid yeast extract agarEMMEdinburgh minimal media. is one of the most widely used methods in quantitative proteomics (1.Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1317) Google Scholar). It involves in vivo metabolic labeling of cell cultures (or small organisms) with different versions of stable isotope-labeled amino acids (2.Ong 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). To maximize the number of peptides that can be quantified after proteome digestion with trypsin, proteins are usually differentially labeled with different forms of lysine and arginine (3.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2807) Google Scholar): l-lysine (Lys-0) and l-arginine (Arg-0); 2H4-lysine(Lys-4) and 13C6-arginine (Arg-6); or 13C6-15N2-lysine (Lys-8) and 13C6-15N4-arginine (Arg-10). The availability of multiple forms of labeled lysine and arginine support the application of SILAC in duplex (comparison of two states) or triplex (comparison of three states) formats. Efficient anabolic pathways mean that lysine and arginine are not essential for growth of wild type yeast cells. Auxotrophic mutants that are defective in these pathways can be used to switch yeast to an absolute dependence upon the provision of these amino acids in the external medium. Consequently, mutations in arginine and lysine biosynthesis pathways can be used to drive the complete labeling of all tryptic peptides with specific forms of these amino acids (4.de Godoy L.M.F. Olsen J.V. de Souza G.A. Li G.Q. Mortensen P. Mann M. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system.Genome Biol. 2006; 7: R50Crossref PubMed Scopus (231) Google Scholar, 5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). SILAC has been used in quantitative proteomics in several yeast species, but most widely in Saccharomyces cerevisiae (6.de Godoy L.M. Olsen J.V. Cox J. Nielsen M.L. Hubner N.C. Frohlich F. Walther T.C. Mann M. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.Nature. 2008; 455: 1251-1254Crossref PubMed Scopus (737) Google Scholar) (budding yeast) and Schizosaccharomyces pombe (fission yeast). S. pombe is extensively exploited to study cell cycle control (7.Elledge S.J. Cell cycle checkpoints: preventing an identity crisis.Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1761) Google Scholar), heterochromatin (8.White S.A. Allshire R.C. RNAi-mediated chromatin silencing in fission yeast.Curr. Top. Microbiol. 2008; 320: 157-183Crossref PubMed Scopus (48) Google Scholar), and differentiation (9.Harigaya Y. Yamamoto M. Molecular mechanisms underlying the mitosis–meiosis decision.Chromosome Res. 2007; 15: 523-537Crossref PubMed Scopus (64) Google Scholar) and is increasingly the subject of large-scale quantitative proteomic studies (10.Marguerat S. Schmidt A. Codlin S. Chen W. Aebersold R. Bahler J. Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells.Cell. 2012; 151: 671-683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 11.Gunaratne J. Schmidt A. Quandt A. Neo S.P. Sarac O.S. Gracia T. Loguercio S. Ahrne E. Xia R.L. Tan K.H. Lossner C. Bahler J. Beyer A. Blackstock W. Aebersold R. Extensive mass spectrometry-based analysis of the fission yeast proteome: the Schizosaccharomyces pombe PeptideAtlas.Mol. Cell. Proteomics. 2013; 12: 1741-1751Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). stable isotope labeling by amino acids in cell culture solid yeast extract agar Edinburgh minimal media. stable isotope labeling by amino acids in cell culture solid yeast extract agar Edinburgh minimal media. A major challenge that is faced when using SILAC in fission yeast, is metabolic conversion of arginine to other amino acids such as proline, glutamine, and lysine (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This partial labeling of additional amino acids after the conversion event produces spectra with complex isotope clusters that makes the downstream analysis challenging and error-prone. Inactivation of the “arginine conversion pathway” by removal of the orthinine transferase, Car2, effectively overcomes this problem to support the use of arginine labeling in SILAC-based experiments (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Although this exploitation of the car2.Δ mutation now enables SILAC technology in fission yeast, the choice of amino acids that can be employed remains limited. Only one form of heavy arginine (R6) is currently used alongside three forms of heavy lysine (Lys-4, Lys-6, and Lys-8) (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12.Koch A. Krug K. Pengelley S. Macek B. Hauf S. Mitotic substrates of the kinase aurora with roles in chromatin regulation identified through quantitative phosphoproteomics of fission yeast.Sci. Signal. 2011; 4: rs6Crossref PubMed Scopus (92) Google Scholar). Surprisingly, we could not find any studies that use arginine (Arg-10) in fission yeast, even though this is a widely exploited reagent for labeling other cell types (1.Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1317) Google Scholar). Here, we show that labeling of fission yeast with Arg-10 leads to a general misincorporation of the stable isotope label that prevents the identification of labeled peptides. We hypothesize that successive arginase and urease activities catabolize the guanidinium group of Arg-10 to 15N1-labeled ammonia. This labeled ammonium is then used as a general precursor for amino acid biosynthesis. Disruption of Ni2+-dependent urease activity through deletion of Ni2+ transporter Nic1 in ammonium-supplemented medium, blocked this recycling to support 13C615N4-arginine labeling SILAC strategies. As a proof of principle we employ Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe. Yeast Strains- Strains used in this used study are listed Table 1. Cell culture and maintenance were according to Moreno et al. (13.Moreno S. Klar A. Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3137) Google Scholar). The amino acid supplements were uracil, adenine, leucine, and histidine at a concentration of 200 μg/ml. Yeast strains were streaked onto YES plates (Solid Yeast Extract agar with supplements) from frozen glycerol stocks at −80 °C, and grown at the permissive temperature (25 °C) until single colonies formed. One single colony was used to start each liquid culture. For all physiological experiments, asynchronous cultures of cells were grown in variants of Edinburgh minimal media 2 (EMM2), with appropriate supplements from log phase starter cultures (1 × 106 – 5 × 106 cells/ml). In EMMG, the 5g/L ammonium chloride of EMM2 is replaced with 5g/L monosodium glutamate (14.Fantes P. Nurse P. Control of cell size at division in fission yeast by a growth-modulated size control over nuclear division.Exp. Cell Res. 1977; 107: 377-386Crossref PubMed Scopus (305) Google Scholar). In EMMGn, EMMG is supplemented with 12 mm NH4Cl.Table IOverview of the cdc25.22 arrest-release study in a that been grown in YES to phase × 106 – × 106 were by at × for 2 in by in appropriate of to a of 2 × at of was to of cells alongside of in at and at cells were in and onto minimal media. and were used as medium and were used as heavy EMMG media is to EMM2 P. control of cell size at cell division in yeast.Nature. PubMed Scopus Google Scholar) that 5 ammonium chloride is replaced with 5 monosodium glutamate (14.Fantes P. Nurse P. Control of cell size at division in fission yeast by a growth-modulated size control over nuclear division.Exp. Cell Res. 1977; 107: 377-386Crossref PubMed Scopus (305) Google Scholar). EMM2 media was with a of ammonium chloride to is EMMG 12 mm To label the cells with heavy or medium of and starter cultures were by supplemented with each of and versions of arginine and from log phase starter cultures were grown in supplemented with of heavy labeled and for at to the labeling of the was that the culture phase (1 – 5 × culture was at For with the EMM2 experiments of et al. (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) was to starter cultures in YES media as we were to growth in cultures of the SILAC when the starter and culture were in minimal medium. The were such that the were to the the and the were to the of the that the P. B. J. for disruption and in Schizosaccharomyces 2005; PubMed Scopus Google Scholar). The was the colonies that were to were analysis the two of to arginine and lysine and were to the SILAC and The were such that the were to the the and the were to the with the (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, P. B. J. for disruption and in Schizosaccharomyces 2005; PubMed Scopus Google Scholar). The was the colonies that were to were analysis the two of to the SILAC the 5 × cells in log phase of growth were at for 2 in Cell were in mm mm mm and mm frozen in liquid and at −80 were by the cells with 200 acid in the of 200 of 2 in mm in a used for at 200 of was to the a at for A was in the of each each was the of an and at for 2 at The was by at for at The was in a to be for concentration was using in mm ammonium was to the to a concentration of mm a at temperature mm in mm was to a concentration of mm a at in the The was then with at for digestion with at the was to a concentration of with and with trypsin at The were to trypsin by or using as K. A. K. N.C. Macek B. of the proteome enables the of in simple Cell. Proteomics. 2013; 12: Full Text Full Text PDF PubMed Scopus Google Scholar) and using J. Mann M. Y. for and of peptides for proteomics using 2007; PubMed Scopus Google Scholar). was as by Krug et al. K. A. K. N.C. Macek B. of the proteome enables the of in simple Cell. Proteomics. 2013; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). were 12 using with a with the A of were until were were with of and in were using a to an or a mass were a with a with were the in A at using a of were then using a or of in at a of 200 The mass were in the were with using a of or was at or with an of For analysis the or most were from the and with at a of and in the The were with the J. Mann M. enables identification mass and 2008; PubMed Scopus Google Scholar) and using the J. A. Olsen J.V. Mann M. a the Res. 2011; PubMed Scopus Google Scholar). The spectra were a the proteome of the S. pombe from The from the S. pombe observed from as as the of all Mass was to at the and at the A minimal of amino acids and tryptic were were of and were as and of was used as The was to the and In quantitative SILAC amino acids were as were as with analysis and were as by et al. A. of a the of to a approach to the study of in fission Cell 2013; PubMed Scopus Google Scholar). The and A. T. R. K. of the of by a of Cell PubMed Google Scholar) were and and were used in as for and at of and was used in To the of using arginine (Arg-10) for quantitative proteomics of fission yeast, we SILAC labeling of S. pombe cells as by et al. (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We used and to this as in the with (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), the SILAC in the EMM2 medium that ammonium as a one of two arginine to the growth of arginine such as the SILAC J. and the of arginine in the fission yeast Schizosaccharomyces Microbiol. Google Scholar). of ammonium for monosodium glutamate in the medium EMMG enables arginine pathways to be phase growth of the SILAC in EMMG was to wild type growth in the medium We cells in EMMG the ammonium EMM2 used by et al. EMMG is widely used the fission yeast an upon growth J. signaling through and kinase in to T. PubMed Scopus Google Scholar), from growth EMM2 been as an additional we the of the SILAC mutations the expression of from of kinase and the 5 a SILAC to A. A. S. A. the and of fission Cell Biol. 2013; 15: PubMed Scopus Google Scholar, I. M. by the fission yeast PubMed Scopus Google Scholar) to an that the switch of the of SILAC any upon the of by these mutations a SILAC experiment using and and and and amino acids in EMMG we that all heavy and peptides complex isotope clusters that effectively identification of heavy peptides and The and peptides in these complex clusters is of a conversion of the stable isotope label of the heavy arginine To the labeling problem in we the and heavy labeled The identification for and labeled was was in from heavy labeled cells. To the heavy amino acid for the we labeled with a single heavy label or Only the heavy arginine the complex isotope clusters that identification at and Arg-10 is with SILAC labeling currently used for fission yeast a of the proteins identified in each experiment is in to We that this of multiple amino acids from of group of heavy arginine (Arg-10) to heavy by and the group that could be catabolized by ammonia (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The from then be multiple amino each different of to the isotope of all peptides. the 13C6-arginine heavy (Arg-10) has labeled and labeled with this form of arginine (Arg-10) are to this urease conversion was not observed in of amino acids arginine and lysine Ni2+ as the major for S. pombe one urease and of urease from Schizosaccharomyces J. Microbiol. 1996; PubMed Scopus Google Scholar) alongside several other proteins that are to urease Nic1 is the sole Ni2+ transporter and urease activity of fission yeast T. M. a of in Schizosaccharomyces for urease Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of et al. T. M. a of in Schizosaccharomyces for urease Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to that the of the urease activity, from of Nic1 the amino acid we observed growth of the SILAC heavy arginine (Arg-10). We in the SILAC to the SILAC labeling as As major upon the growth of wild type strains the growth of SILAC and strains were that removal of is to cell the mutations that are currently exploited for SILAC labeling in fission yeast (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). of the identification of arginine that arginine was a in arginine (Arg-10) the number of peptides that could be identified in labeled with heavy amino acids was that in amino the of peptides was a partial of This that of Ni2+ in or that the is catabolized to ammonium by Ni2+ The identification of (Arg-10) labeled peptides in the has not been by removal of Nic1, been We we could the of the cells to these of in amino acid anabolic by the EMMG growth medium with of that are by SILAC strains (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The with a of in EMMG media that 12 mm supplemented with forms of lysine and This is to the 5 of the SILAC and of wild type in the medium. stable isotope-labeled amino acids were used as the sole of lysine and arginine the the to at For that are not the SILAC in the isotope of the that growth of cells in the problem spectra with This to of heavy labeled peptides to that are in cells labeled with or medium amino acids yeast is widely exploited for analysis of the cell division We the of the and medium upon the to use mutations to through the cell division of the of the through the mutation cell cycle in cells are to J. C. A. and from of for the start in the fission yeast Schizosaccharomyces Cell PubMed Google Scholar). As phase is after the of cells in an asynchronous culture complete at the G1 P. P. K. control of the cell division cycle in the fission yeast Schizosaccharomyces PubMed Scopus Google Scholar). cell cycle at the is a commonly employed approach to through after the to that is by the to the permissive We this approach could be used to phase in a log phase culture was to to a of cells and an of cells to of 5 through temperature of the mutation the of the two to Consequently, the that are to the are not This in a of the that blocks cell cycle and cells to in with I. M. by the fission yeast PubMed Scopus Google Scholar). The mutation blocked in EMMGn, of the of the mutations Finally, we the of the mutations and growth in upon the widely exploited of the cdc25.22 mutation to cell cycle at the cdc25.22 cultures are with to (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, A. of a the of to a approach to the study of in fission Cell 2013; PubMed Scopus Google Scholar). cdc25.22 cells were grown to log phase (1 × in at a to for The of the that cells an cell cycle at the not The of and in analysis of after to the permissive temperature of an to cycle after We that the of the mutations growth in has a upon the to cell cycle to at cell cycle or with the or cdc25.22 The growth of strains with is yeast This growth major upon the of experiments all three strains the genetic To this approach we a the cdc25.22 experiment by et al. (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this we cdc25.22 cells in supplemented with arginine and lysine for or labeling. were at the to °C, to the analysis of cell cycle in all Because the of the mutation the of triple-SILAC we supplemented the at G2 and by et with a in phase for the at which each was In the were labeled as G2 (Lys-4, and in the experiment we a label (Lys-4, and G2 of the experiment can be in Table I. In we spectra that identified a of peptides from S. pombe at of at the Table As in Table the use of Arg-10 not identification and the number of quantified and were that Arg-10 can be used in SILAC in the cdc25.22 the quantified were quantified in and was in of the that the were in We to the by et al. (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and observed proteins that were in this was in the with SILAC and Arg-10 labeling was a of proteins in we could several observed by et al. (5.Bicho C.C. Alves F.D. Chen Z.A. Rappsilber J. Sawin K.E. A genetic engineering solution to the “arginine conversion problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).Mol. Cell. Proteomics. 2010; 9: 1567-1577Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, A. Krug K. S. A. S. Hauf S. Macek B. proteome and dynamics the cell cycle of fission Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). In to several differentially metabolic we in the of kinase and the complex in the We regulation of the cell cycle the for the cell and the kinase in the and We that removal of Nic1 from cells grown in fission yeast with SILAC to the for of the S. pombe The to SILAC analysis in this model quantitative of of cell We M. Mann and S. Hauf for the as as K. Krug and C. for with of We K. for with

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