A label-free mass spectrometry method for the quantification of protein isotypes.
Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as "built-in" internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from betaI-, betaII-, betaIII-, betaIVb-, and betaV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the beta-tubulin isotypes. To show the utility of this method, we determined that betaI-tubulin represented 77% and betaIII-tubulin represented 3.2% of the total HeLa beta-tubulin.
Winefield
Robert D
RD
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.
Williams
Todd D
TD
Himes
Richard H
RH
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; D020033:Protein Isoforms; D014404:Tubulin; D014357:Trypsin
Intern Med. 2009;48(4):203-8
19218769
Cell Motil Cytoskeleton. 2003 Jun;55(2):77-96
12740870
Anal Biochem. 1976 May 7;72:248-54
942051
Anal Chem. 2008 Feb 15;80(4):1290-6
18217771
Anal Biochem. 2009 Jan 15;384(2):197-206
18840397
Mol Cell Proteomics. 2005 Oct;4(10):1487-502
15979981
Anal Chem. 2001 Jul 1;73(13):2836-42
11467524
Nat Biotechnol. 1999 Oct;17(10):994-9
10504701
Drug Metab Dispos. 2005 Sep;33(9):1399-407
15951447
Plant J. 2008 Sep;55(6):1039-46
18485062
Clin Cancer Res. 2007 Feb 1;13(3):994-9
17289895
Biochemistry. 2003 Oct 21;42(41):12019-27
14556633
Int Rev Cytol. 1998;178:207-75
9348671
Anal Bioanal Chem. 2007 Oct;389(4):1017-31
17668192
Nature. 1998 Jan 8;391(6663):199-203
9428769
J Proteome Res. 2002 Jul-Aug;1(4):317-23
12645887
Plant Cell. 2007 Nov;19(11):3339-46
18055608
Yeast. 2005 Sep;22(12):971-8
16134117
Mol Cancer Ther. 2009 Jan;8(1):17-25
19139109
Cancer Res. 2008 Nov 1;68(21):8881-8
18974132
Proteomics. 2008 Feb;8(4):731-49
18297651
Biochim Biophys Acta. 2001;1471(2):O1-9
11342188
Nature. 2003 Mar 13;422(6928):198-207
12634793
J Biol Chem. 2005 Apr 1;280(13):12902-7
15695826
Proc Natl Acad Sci U S A. 2003 May 27;100(11):6394-7
12740436
Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):6940-5
12771378
Mol Cancer Ther. 2005 Dec;4(12):2001-7
16373715
Genomics. 2007 Aug;90(2):285-9
17543498
Mol Syst Biol. 2007;3:79
17299416
IUBMB Life. 2008 Mar;60(3):165-70
18380008
Mol Cell Proteomics. 2004 Dec;3(12):1154-69
15385600
Anal Chem. 2002 Sep 15;74(18):4741-9
12349978
J Biol Chem. 1998 May 15;273(20):12612-22
9575223
Cancer Res. 2007 Oct 1;67(19):9356-63
17909044
J Biol Chem. 2002 Nov 8;277(45):42456-62
12207023
Nat Chem Biol. 2005 Oct;1(5):252-62
16408053
Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6591-6
10359756