Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.
Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate/expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phosphorylation stoichiometries. By using the developed methods, we can normalize to robustly account for run-to-run variations and variations in amounts of starting material. This procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies peptides that are suitable for normalization purposes. Also, we can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest. Absolute phosphorylation stoichiometry are measured by monitoring the ion currents of a phosphopeptide and its unmodified cognate as the signal intensity changes of both peptide species are correlated. The method is applicable to multiply phosphorylated species (for which one more sample with varying phosphorylation stoichiometry than number of phosphorylation sites is required to correct for the differences in the ionization/detection efficiencies of the phosphopeptide, its partially phosphorylated and unphosphorylated cognates). Last, we can quantitate species with ragged ends resulting from incomplete proteolysis and measure phosphorylation stoichiometries of single samples by controlled dephosphorylation. These approaches were validated and subsequently applied to the phosphorylation of the yeast transcription factor Pho4.
Steen
Hanno
H
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Jebanathirajah
Judith A
JA
Springer
Michael
M
Kirschner
Marc W
MW
D002623:Chemistry Techniques, Analytical; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
D004268:DNA-Binding Proteins; C060292:PHO4 protein, S cerevisiae; D010446:Peptide Fragments; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6591-6
10359756
Anal Chem. 2003 Dec 1;75(23):6658-65
14640742
Anal Chem. 2001 Apr 1;73(7):1440-8
11321292
Rapid Commun Mass Spectrom. 2002;16(24):2325-32
12478578
J Am Soc Mass Spectrom. 2004 May;15(5):647-53
15121193
Anal Chem. 1996 Mar 1;68(5):850-8
8779443
Anal Chem. 2003 May 15;75(10):2370-6
12918979
Mol Cell Proteomics. 2003 Nov;2(11):1234-43
14506206
Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):880-5
12540831
Rapid Commun Mass Spectrom. 2000;14(18):1677-81
10962490
Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5
15302935
Anal Chem. 2001 Jun 1;73(11):2578-86
11403303
J Mol Biol. 2001 Mar 9;306(5):997-1010
11237614
J Chromatogr A. 1998 May 29;808(1-2):23-41
9652109
Nature. 2003 Dec 4;426(6966):570-4
14654843
Anal Biochem. 1996 Aug 1;239(2):180-92
8811904
Anal Chem. 2003 Feb 1;75(3):663-70
12585499
Anal Chem. 2003 Nov 15;75(22):6043-9
14615979
Philos Trans R Soc Lond B Biol Sci. 1998 Apr 29;353(1368):583-605
9602534
Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):6940-5
12771378
Anal Chem. 2003 Oct 15;75(20):5441-50
14714534
Anal Chem. 2002 Apr 1;74(7):1658-64
12033257
Anal Chem. 1999 Jul 15;71(14):2883-92
10424175
Anal Chem. 2003 Sep 15;75(18):4818-26
14674459
J Biomol Tech. 2002 Sep;13(3):119-30
19498976
Anal Chem. 2002 Sep 15;74(18):4741-9
12349978