A genetic engineering solution to the "arginine conversion problem" in stable isotope labeling by amino acids in cell culture (SILAC).
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.
Bicho
Claudia C
CC
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
de Lima Alves
Flavia
F
Chen
Zhuo A
ZA
Rappsilber
Juri
J
Sawin
Kenneth E
KE
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
D000596:Amino Acids; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D001120:Arginine
Microbiol Rev. 1986 Sep;50(3):280-313
2945985
Mol Cell Proteomics. 2008 Sep;7(9):1587-97
18487603
Nat Protoc. 2006;1(6):2650-60
17406521
Nature. 1996 May 30;381(6581):438-41
8632802
Mol Biol Cell. 2005 Mar;16(3):1378-95
15647375
Mol Biosyst. 2009 Nov;5(11):1337-46
19823750
Nat Protoc. 2007;2(8):1896-906
17703201
Nature. 2002 Jul 25;418(6896):387-91
12140549
Science. 1997 Feb 28;275(5304):1311-4
9036857
Nat Methods. 2007 Sep;4(9):677-8
17762871
Nucleic Acids Res. 2010 Jan;38(Database issue):D699-702
19934261
Mol Biol Cell. 2008 Apr;19(4):1727-38
18256290
EMBO J. 2001 Mar 15;20(6):1259-70
11250892
Biosci Rep. 2002 Oct-Dec;22(5-6):487-99
12635846
Cell. 2008 Jul 25;134(2):353-64
18662549
J Cell Sci. 2000 Nov;113 ( Pt 22):3989-99
11058086
Mol Biol Cell. 2005 Apr;16(4):1756-68
15689489
Yeast. 1998 Jul;14(10):943-51
9717240
Rapid Commun Mass Spectrom. 2007;21(23):3919-26
17979108
Methods Enzymol. 1991;194:795-823
2005825
J Proteome Res. 2008 Mar;7(3):1088-97
18257517
Nat Biotechnol. 2005 May;23(5):617-21
15834404
Cell. 2009 Aug 21;138(4):795-806
19664813
EMBO J. 1996 Oct 1;15(19):5268-79
8895572
Nat Methods. 2010 May;7(5):383-5
20364148
Mol Biol Cell. 2005 Mar;16(3):1026-42
15616197
Nature. 2008 Oct 30;455(7217):1251-4
18820680
Yeast. 1994 Jul;10(7):923-33
7985419
PLoS Biol. 2005 Jul;3(7):e225
15966770
J Gen Microbiol. 1984 Dec;130(12):3265-73
18357653
Nature. 2008 Jun 26;453(7199):1239-43
18488015
Nat Biotechnol. 2008 Dec;26(12):1367-72
19029910
Mol Cell Proteomics. 2008 May;7(5):845-52
18087068
Nat Genet. 2004 Aug;36(8):809-17
15195092
J Proteome Res. 2003 Mar-Apr;2(2):173-81
12716131
Mol Cell Proteomics. 2005 Mar;4(3):310-27
15665377
Mol Cell Proteomics. 2006 Jun;5(6):1131-45
16540461
J Chromatogr A. 2002 Dec 6;979(1-2):233-9
12498253
Mol Cell Proteomics. 2009 May;8(5):959-70
19151416
J Cell Sci. 2003 May 1;116(Pt 9):1689-98
12665550
Nat Methods. 2009 Mar;6(3):184-5
19247291
Cell Physiol Biochem. 2000;10(1-2):37-55
10846151
Methods. 2006 Nov;40(3):243-50
17071406
Mol Syst Biol. 2007;3:79
17299416
Methods. 2009 Jul;48(3):311-9
19272449
BMC Genomics. 2003 Jul 10;4(1):27
12854975
Cell. 1989 Jul 28;58(2):361-72
2665944
Cell Biochem Biophys. 2006;46(1):43-64
16943623
Yeast. 2006 Mar;23(4):261-277
16544289
Anal Chem. 1996 Mar 1;68(5):850-8
8779443
Mol Cell Proteomics. 2002 May;1(5):376-86
12118079
J Cell Sci. 2004 Nov 1;117(Pt 23):5623-32
15509866
Cell. 1989 Aug 11;58(3):485-97
2569363
Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15281-6
18829439
J Biol Chem. 1995 Mar 31;270(13):7703-11
7706319
J Cell Biol. 2003 Mar 31;160(7):1093-103
12668659
Nat Chem Biol. 2005 Oct;1(5):252-62
16408053
Mol Cell Proteomics. 2008 Apr;7(4):672-83
18045802
Curr Biol. 2004 May 4;14(9):763-75
15120067
Anal Chem. 2003 Feb 1;75(3):663-70
12585499