title: Stable isotope labeling tandem mass spectrometry (SILT): integration with peptide identification and extension to data-dependent scans.
abstract: Quantitation of relative or absolute amounts of proteins by mass spectrometry can be prone to large errors. The use of MS/MS ion intensities and stable isotope labeling, which we term stable isotope labeling tandem mass spectrometry (SILT), decreases the effects of contamination from unrelated compounds. We present a software package (SILTmass) that automates protein identification and quantification by the SILT method. SILTmass has the ability to analyze the kinetics of protein turnover, in addition to relative and absolute protein quantitation. Instead of extracting chromatograms to find elution peaks, SILTmass uses only scans in which a peptide is identified and that meet an ion intensity threshold. Using only scans with identified peptides, the accuracy and precision of SILT is shown to be superior to precursor ion intensities, particularly at high or low dilutions of the isotope labeled compounds or with low amounts of protein. Using example scans, we demonstrate likely reasons for the improvements in quantitation by SILT. The appropriate use of variable modifications in peptide identification is described for measurement of protein turnover kinetics. The combination of identification with SILT facilitates quantitation without peak detection and helps to ensure the appropriate use of variable modifications for kinetics experiments.
authors.affiliation: Department of Biomedical Engineering and Center for Materials Innovation, Washington University, St. Louis, Missouri, USA. elbert@biomed.wustl.edu
authors.forename: Donald L
authors.initials: DL
authors.lastname: Elbert
authors.forename: Kwasi G
authors.initials: KG
authors.lastname: Mawuenyega
authors.forename: Evan A
authors.initials: EA
authors.lastname: Scott
authors.forename: Kristin R
authors.initials: KR
authors.lastname: Wildsmith
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Bateman
mesh_terms: D000818:Animals; D001057:Apolipoproteins E / Q000032:analysis / Q000235:genetics; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D010455:Peptides / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001057:Apolipoproteins E; D010455:Peptides
references.citation: J Biomed Mater Res A. 2005 Oct 1;75(1):199-209
references.pmid: 16082704
references.citation: Methods. 2003 Feb;29(2):124-30
references.pmid: 12606218
references.citation: Clin Chem. 2003 Jul;49(7):1041-4
references.pmid: 12816898
references.citation: Proteomics. 2002 Oct;2(10):1406-12
references.pmid: 12422357
references.citation: Nat Biotechnol. 1999 Oct;17(10):994-9
references.pmid: 10504701
references.citation: Anal Chem. 2004 Jul 15;76(14):4193-201
references.pmid: 15253663
references.citation: Mol Cell Proteomics. 2007 Aug;6(8):1291-8
references.pmid: 17513294
references.citation: Proteomics. 2004 Aug;4(8):2333-51
references.pmid: 15274127
references.citation: Nat Med. 2006 Jul;12(7):856-61
references.pmid: 16799555
references.citation: Methods. 2005 Mar;35(3):265-73
references.pmid: 15722223
references.citation: Curr Opin Lipidol. 2003 Feb;14(1):61-8
references.pmid: 12544663
references.citation: Electrophoresis. 1999 Dec;20(18):3551-67
references.pmid: 10612281
references.citation: J Am Soc Mass Spectrom. 1994 Nov;5(11):976-89
references.pmid: 24226387
references.citation: Mol Cell Proteomics. 2006 Feb;5(2):401-11
references.pmid: 16253985
references.citation: Proc Natl Acad Sci U S A. 1982 Feb;79(4):1163-5
references.pmid: 6951165
references.citation: Am J Physiol. 1992 Nov;263(5 Pt 1):E988-1001
references.pmid: 1443132
references.citation: J Proteome Res. 2004 May-Jun;3(3):350-63
references.pmid: 15253416
references.citation: Neurobiol Dis. 2005 Jun-Jul;19(1-2):66-76
references.pmid: 15837562
references.citation: J Am Soc Mass Spectrom. 2007 Jun;18(6):997-1006
references.pmid: 17383190
references.citation: Nat Biotechnol. 2004 Nov;22(11):1459-66
references.pmid: 15529173
references.citation: Am J Physiol. 1999 Jun;276(6):E1146-70
references.pmid: 10362629
references.citation: Anal Chem. 1999 Jul 15;71(14):2871-82
references.pmid: 10424174
references.citation: Nat Methods. 2004 Oct;1(1):39-45
references.pmid: 15782151
references.citation: Anal Biochem. 1999 Feb 1;267(1):1-16
references.pmid: 9918649
references.citation: Nat Methods. 2008 Apr;5(4):319-22
references.pmid: 18345006
references.citation: Biomaterials. 2007 Sep;28(27):3904-17
references.pmid: 17582492
references.citation: Mol Cell Proteomics. 2004 Dec;3(12):1154-69
references.pmid: 15385600
references.citation: J Proteome Res. 2007 Oct;6(10):3962-75
references.pmid: 17711321
references.citation: Mol Cell Proteomics. 2002 Feb;1(2):148-56
references.pmid: 12096133
references.citation: Mol Cell Proteomics. 2002 May;1(5):376-86
references.pmid: 12118079
references.citation: Nat Chem Biol. 2005 Oct;1(5):252-62
references.pmid: 16408053
references.citation: J Biol Chem. 2007 Aug 10;282(32):23427-36
references.pmid: 17567584
references.citation: Proteomics. 2002 Jan;2(1):36-47
references.pmid: 11788990
references.citation: J Biol Chem. 2000 Aug 25;275(34):26277-84
references.pmid: 10816583
references.citation: J Proteome Res. 2004 Sep-Oct;3(5):958-64
references.pmid: 15473683
references.citation: Electrophoresis. 1998 May;19(6):998-1005
references.pmid: 9638946
references.citation: Proteomics. 2005 Jan;5(1):4-15
references.pmid: 15602776
references.citation: Anal Chem. 2003 Dec 1;75(23):6648-57
references.pmid: 14640741