title: A genetic engineering solution to the "arginine conversion problem" in stable isotope labeling by amino acids in cell culture (SILAC).
abstract: Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the "arginine conversion problem" may be more broadly applicable to organisms amenable to genetic manipulation.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
authors.forename: Claudia C
authors.initials: CC
authors.lastname: Bicho
authors.forename: Flavia
authors.initials: F
authors.lastname: de Lima Alves
authors.forename: Zhuo A
authors.initials: ZA
authors.lastname: Chen
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Sawin
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism
chemical_list: D000596:Amino Acids; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D001120:Arginine
references.citation: Microbiol Rev. 1986 Sep;50(3):280-313
references.pmid: 2945985
references.citation: Mol Cell Proteomics. 2008 Sep;7(9):1587-97
references.pmid: 18487603
references.citation: Nat Protoc. 2006;1(6):2650-60
references.pmid: 17406521
references.citation: Nature. 1996 May 30;381(6581):438-41
references.pmid: 8632802
references.citation: Mol Biol Cell. 2005 Mar;16(3):1378-95
references.pmid: 15647375
references.citation: Mol Biosyst. 2009 Nov;5(11):1337-46
references.pmid: 19823750
references.citation: Nat Protoc. 2007;2(8):1896-906
references.pmid: 17703201
references.citation: Nature. 2002 Jul 25;418(6896):387-91
references.pmid: 12140549
references.citation: Science. 1997 Feb 28;275(5304):1311-4
references.pmid: 9036857
references.citation: Nat Methods. 2007 Sep;4(9):677-8
references.pmid: 17762871
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D699-702
references.pmid: 19934261
references.citation: Mol Biol Cell. 2008 Apr;19(4):1727-38
references.pmid: 18256290
references.citation: EMBO J. 2001 Mar 15;20(6):1259-70
references.pmid: 11250892
references.citation: Biosci Rep. 2002 Oct-Dec;22(5-6):487-99
references.pmid: 12635846
references.citation: Cell. 2008 Jul 25;134(2):353-64
references.pmid: 18662549
references.citation: J Cell Sci. 2000 Nov;113 ( Pt 22):3989-99
references.pmid: 11058086
references.citation: Mol Biol Cell. 2005 Apr;16(4):1756-68
references.pmid: 15689489
references.citation: Yeast. 1998 Jul;14(10):943-51
references.pmid: 9717240
references.citation: Rapid Commun Mass Spectrom. 2007;21(23):3919-26
references.pmid: 17979108
references.citation: Methods Enzymol. 1991;194:795-823
references.pmid: 2005825
references.citation: J Proteome Res. 2008 Mar;7(3):1088-97
references.pmid: 18257517
references.citation: Nat Biotechnol. 2005 May;23(5):617-21
references.pmid: 15834404
references.citation: Cell. 2009 Aug 21;138(4):795-806
references.pmid: 19664813
references.citation: EMBO J. 1996 Oct 1;15(19):5268-79
references.pmid: 8895572
references.citation: Nat Methods. 2010 May;7(5):383-5
references.pmid: 20364148
references.citation: Mol Biol Cell. 2005 Mar;16(3):1026-42
references.pmid: 15616197
references.citation: Nature. 2008 Oct 30;455(7217):1251-4
references.pmid: 18820680
references.citation: Yeast. 1994 Jul;10(7):923-33
references.pmid: 7985419
references.citation: PLoS Biol. 2005 Jul;3(7):e225
references.pmid: 15966770
references.citation: J Gen Microbiol. 1984 Dec;130(12):3265-73
references.pmid: 18357653
references.citation: Nature. 2008 Jun 26;453(7199):1239-43
references.pmid: 18488015
references.citation: Nat Biotechnol. 2008 Dec;26(12):1367-72
references.pmid: 19029910
references.citation: Mol Cell Proteomics. 2008 May;7(5):845-52
references.pmid: 18087068
references.citation: Nat Genet. 2004 Aug;36(8):809-17
references.pmid: 15195092
references.citation: J Proteome Res. 2003 Mar-Apr;2(2):173-81
references.pmid: 12716131
references.citation: Mol Cell Proteomics. 2005 Mar;4(3):310-27
references.pmid: 15665377
references.citation: Mol Cell Proteomics. 2006 Jun;5(6):1131-45
references.pmid: 16540461
references.citation: J Chromatogr A. 2002 Dec 6;979(1-2):233-9
references.pmid: 12498253
references.citation: Mol Cell Proteomics. 2009 May;8(5):959-70
references.pmid: 19151416
references.citation: J Cell Sci. 2003 May 1;116(Pt 9):1689-98
references.pmid: 12665550
references.citation: Nat Methods. 2009 Mar;6(3):184-5
references.pmid: 19247291
references.citation: Cell Physiol Biochem. 2000;10(1-2):37-55
references.pmid: 10846151
references.citation: Methods. 2006 Nov;40(3):243-50
references.pmid: 17071406
references.citation: Mol Syst Biol. 2007;3:79
references.pmid: 17299416
references.citation: Methods. 2009 Jul;48(3):311-9
references.pmid: 19272449
references.citation: BMC Genomics. 2003 Jul 10;4(1):27
references.pmid: 12854975
references.citation: Cell. 1989 Jul 28;58(2):361-72
references.pmid: 2665944
references.citation: Cell Biochem Biophys. 2006;46(1):43-64
references.pmid: 16943623
references.citation: Yeast. 2006 Mar;23(4):261-277
references.pmid: 16544289
references.citation: Anal Chem. 1996 Mar 1;68(5):850-8
references.pmid: 8779443
references.citation: Mol Cell Proteomics. 2002 May;1(5):376-86
references.pmid: 12118079
references.citation: J Cell Sci. 2004 Nov 1;117(Pt 23):5623-32
references.pmid: 15509866
references.citation: Cell. 1989 Aug 11;58(3):485-97
references.pmid: 2569363
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15281-6
references.pmid: 18829439
references.citation: J Biol Chem. 1995 Mar 31;270(13):7703-11
references.pmid: 7706319
references.citation: J Cell Biol. 2003 Mar 31;160(7):1093-103
references.pmid: 12668659
references.citation: Nat Chem Biol. 2005 Oct;1(5):252-62
references.pmid: 16408053
references.citation: Mol Cell Proteomics. 2008 Apr;7(4):672-83
references.pmid: 18045802
references.citation: Curr Biol. 2004 May 4;14(9):763-75
references.pmid: 15120067
references.citation: Anal Chem. 2003 Feb 1;75(3):663-70
references.pmid: 12585499