A robust method for quantitative high-throughput analysis of proteomes by 18O labeling.
MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of (18)O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide (18)O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with (18)O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by (18)O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail.
Bonzon-Kulichenko
Elena
E
Laboratory of Protein Chemistry and Proteomics, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, E-28049, Spain.
Pérez-Hernández
Daniel
D
Núñez
Estefanía
E
Martínez-Acedo
Pablo
P
Navarro
Pedro
P
Trevisan-Herraz
Marco
M
Ramos
María del Carmen
Mdel C
Sierra
Saleta
S
Martínez-Martínez
Sara
S
Ruiz-Meana
Marisol
M
Miró-Casas
Elizabeth
E
García-Dorado
David
D
Redondo
Juan Miguel
JM
Burgos
Javier S
JS
Vázquez
Jesús
J
D000704:Analysis of Variance; D000818:Animals; D045744:Cell Line, Tumor; D005591:Chemical Fractionation; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing; D007553:Isotope Labeling / Q000379:methods*; D008715:Methionine / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D051381:Rats
D009363:Neoplasm Proteins; D010103:Oxygen Isotopes; D010455:Peptides; D020543:Proteome; D008715:Methionine
J Biol Chem. 2006 Nov 3;281(44):33078-86
16945918
Proteomics. 2006 Apr;6 Suppl 1:S16-27
16534747
Mol Cell Proteomics. 2006 Oct;5(10):1968-74
16849286
Nature. 2008 Oct 30;455(7217):1251-4
18820680
Rapid Commun Mass Spectrom. 2004;18(20):2388-94
15386632
Electrophoresis. 1996 May;17(5):945-53
8783021
Mol Biol Cell. 2005 Mar;16(3):1543-54
15647377
J Proteome Res. 2007 Jan;6(1):263-72
17203970
Nat Biotechnol. 2001 Mar;19(3):242-7
11231557
J Proteome Res. 2009 Apr;8(4):1792-6
19714873
J Proteome Res. 2004 Jul-Aug;3(4):786-91
15359732
J Biol Chem. 2005 May 27;280(21):20867-78
15743762
Proteomics. 2005 May;5(7):1742-5
15761957
J Immunol. 2008 Aug 1;181(3):1927-36
18641330
Mol Cell Biochem. 2005 Jan;269(1-2):85-94
15786719
J Mass Spectrom. 2007 Nov;42(11):1391-403
17960563
Electrophoresis. 2004 Mar;25(6):936-45
15004858
Rapid Commun Mass Spectrom. 2000;14(14):1226-32
10918372
Nat Protoc. 2006;1(6):3019-27
17406563
Proteomics. 2006 Apr;6 Suppl 1:S4-11
16534745
Nat Immunol. 2001 Apr;2(4):316-24
11276202
J Virol. 1990 Mar;64(3):1335-44
2406459
J Proteome Res. 2007 Nov;6(11):4200-9
17902639
Proteomics. 2007 Sep;7(18):3404-16
17708596
Anal Chem. 2001 Jul 1;73(13):2836-42
11467524
Circ Res. 2009 Feb 13;104(3):328-36
19106412
J Proteome Res. 2005 Nov-Dec;4(6):2126-32
16335958
J Biol Chem. 1996 Nov 29;271(48):30417-25
8940006
Mol Cell Proteomics. 2009 May;8(5):1130-49
19181660
J Proteome Res. 2005 Mar-Apr;4(2):523-31
15822930
Nat Biotechnol. 1999 Oct;17(10):994-9
10504701
J Cell Sci. 2010 Apr 1;123(Pt 7):1160-70
20215400
Proteome Sci. 2008 Feb 26;6:9
18302743
Mol Cell Proteomics. 2002 May;1(5):376-86
12118079
Mol Cell Proteomics. 2008 May;7(5):853-63
17951628
Mol Cell Proteomics. 2007 Jul;6(7):1274-86
17322307
Nat Protoc. 2006;1(6):2856-60
17406544
Mol Cell Proteomics. 2007 Jun;6(6):953-62
17296599
Physiol Genomics. 2007 Oct 22;31(2):357-63
17684036
J Proteome Res. 2005 Nov-Dec;4(6):2273-82
16335976
Anal Chem. 2004 Dec 1;76(23):6853-60
15571333
Nat Methods. 2009 May;6(5):359-62
19377485
Nat Methods. 2009 Nov;6(11):785; author reply 785-6
19876013
Proteomics. 2010 Aug;10(15):2758-68
20486122
Mol Cell Proteomics. 2008 Jun;7(6):1135-45
18303013
Apoptosis. 2005 Dec;10(6):1469-82
16215688
Rapid Commun Mass Spectrom. 2004;18(8):869-76
15095355
EMBO Rep. 2009 Dec;10(12):1341-7
19820692
Mol Cell Proteomics. 2008 Apr;7(4):672-83
18045802
Genome Biol. 2006;7(6):R50
16784548
Anal Chem. 2003 Dec 1;75(23):6648-57
14640741
Cardiovasc Res. 2005 Aug 1;67(2):234-44
15919068
Biochim Biophys Acta. 2003 Sep 23;1642(1-2):87-96
12972297
Proc Natl Acad Sci U S A. 2008 Jun 17;105(24):8197-202
18287020
Eur J Immunol. 2002 Nov;32(11):3225-34
12555668