A systematic approach to the analysis of protein phosphorylation
- PMID: 11283598
- DOI: 10.1038/86777
A systematic approach to the analysis of protein phosphorylation
Abstract
Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography-tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture.
Comment in
-
Toward the phosphoproteome.Nat Biotechnol. 2001 Apr;19(4):317-8. doi: 10.1038/86687. Nat Biotechnol. 2001. PMID: 11283582 No abstract available.
Similar articles
-
Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching.Biochemistry. 1998 Nov 17;37(46):16211-24. doi: 10.1021/bi981264p. Biochemistry. 1998. PMID: 9819213
-
Phosphatase-directed phosphorylation-site determination: a synthesis of methods for the detection and identification of phosphopeptides.J Proteome Res. 2005 Sep-Oct;4(5):1628-35. doi: 10.1021/pr050129d. J Proteome Res. 2005. PMID: 16212415
-
Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis.J Proteome Res. 2007 May;6(5):1812-21. doi: 10.1021/pr060631d. Epub 2007 Apr 3. J Proteome Res. 2007. PMID: 17402769
-
Methods in enzymology: O-glycosylation of proteins.Methods Enzymol. 2005;405:139-71. doi: 10.1016/S0076-6879(05)05007-X. Methods Enzymol. 2005. PMID: 16413314 Review.
-
Tools for analyzing the phosphoproteome and other phosphorylated biomolecules: a review.Anal Chim Acta. 2011 Oct 3;703(1):19-30. doi: 10.1016/j.aca.2011.07.012. Epub 2011 Jul 19. Anal Chim Acta. 2011. PMID: 21843671 Review.
Cited by
-
MILKSHAKE Western blot and Sundae ELISA: We all scream for better antibody validation.J Immunol Methods. 2023 Oct;521:113540. doi: 10.1016/j.jim.2023.113540. Epub 2023 Aug 18. J Immunol Methods. 2023. PMID: 37597727
-
Regulation of autophagy and lipid accumulation under phosphate limitation in Rhodotorula toruloides.Front Microbiol. 2023 Jan 26;13:1046114. doi: 10.3389/fmicb.2022.1046114. eCollection 2022. Front Microbiol. 2023. PMID: 36777022 Free PMC article.
-
Magnetic mesoporous silica nanoparticles modified by phosphonate functionalized ionic liquid for selective enrichment of phosphopeptides.RSC Adv. 2022 Sep 21;12(41):26859-26865. doi: 10.1039/d2ra04609a. eCollection 2022 Sep 16. RSC Adv. 2022. PMID: 36320858 Free PMC article.
-
Covalent Chemical Tools for Profiling Post-Translational Modifications.Front Chem. 2022 Jul 4;10:868773. doi: 10.3389/fchem.2022.868773. eCollection 2022. Front Chem. 2022. PMID: 35860626 Free PMC article. Review.
-
Current Methods of Post-Translational Modification Analysis and Their Applications in Blood Cancers.Cancers (Basel). 2021 Apr 16;13(8):1930. doi: 10.3390/cancers13081930. Cancers (Basel). 2021. PMID: 33923680 Free PMC article. Review.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases