Mass Spectrometric Contributions to the Practice of Phosphorylation Site Mapping through 2003

Reversible phosphorylation of proteins is among the most important post-translational modifications, and elucidation of sites of phosphorylation is essential to understanding the regulation of key cellular processes such as signal transduction. Unfortunately phosphorylation site mapping is as technically challenging as it is important. Limitations in the traditional method of Edman degradation of 32P-labeled phosphoproteins have spurred the development of mass spectrometric methods for phosphopeptide identification and sequencing. To assess the practical contributions of the various technologies we conducted a literature search of publications using mass spectrometry to discover previously unknown phosphorylation sites. 1281 such phosphorylation sites were reported in 203 publications between 1992 and 2003. This review examines and catalogues those methods, identifies the trends that have emerged in the past decade, and presents representative examples from among these methods. Reversible phosphorylation of proteins is among the most important post-translational modifications, and elucidation of sites of phosphorylation is essential to understanding the regulation of key cellular processes such as signal transduction. Unfortunately phosphorylation site mapping is as technically challenging as it is important. Limitations in the traditional method of Edman degradation of 32P-labeled phosphoproteins have spurred the development of mass spectrometric methods for phosphopeptide identification and sequencing. To assess the practical contributions of the various technologies we conducted a literature search of publications using mass spectrometry to discover previously unknown phosphorylation sites. 1281 such phosphorylation sites were reported in 203 publications between 1992 and 2003. This review examines and catalogues those methods, identifies the trends that have emerged in the past decade, and presents representative examples from among these methods. Protein phosphorylation is vital to a host of protein functions that are important to cellular processes spanning the range from signal transduction, differentiation, and development to cell cycle control and metabolism. A primary role of phosphorylation is to act as a switch to turn “on” or “off” a protein activity or a cellular pathway in an acute and reversible manner (1.Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling.Cell. 1995; 80: 225-236Google Scholar). Sequencing of the human genome has highlighted the physiological importance of phosphorylation. Kinases and phosphatases, the enzymes responsible for protein phosphorylation and dephosphorylation, respectively, make up ∼2% of the human genome (2.Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. Zhang Q. Kodira C.D. Zheng X.H. Chen L. Skupski M. 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A analysis of mass in the past for the analysis and of phosphorylation as in that a of methods have and in for the phosphorylation analysis of The and of methods in the literature for mass phosphorylation site the a of to the of mass spectrometry has the of is to from the literature of these methods is in we a of methods to have for the of previously unknown protein phosphorylation sites. that the of to the methods that have for phosphorylation site the methods to phosphorylation sites methods have in the literature have for phosphorylation site or have of for of in protein phosphorylation analysis as The literature the and from the 1992 2003. is that were to of these in or in for were that the of phosphorylation sites in mass spectrometry a role in and those that of or sites were A of 203 from 1992 to in and were to these and the phosphoproteins and methods in are in and The were of methods in phosphorylation mapping those methods the or of the in or in phosphorylation were of mass and of phosphorylation or as as sites were of the have as and in phosphorylation site or in a and in phosphorylation site The of that have of the methods or in phosphorylation site is for the and and in site to the of using mass phosphorylation site identification methods for the The of mass spectrometry in protein phosphorylation site has in the past in of that mass spectrometry to protein phosphorylation sites from the from the from and from the decade, it is that in most in have for protein phosphorylation site analysis and that of to for the methods as for phosphorylation site analysis in methods in the search for analysis mass spectrometry is a The of is and the are to are those are mass spectrometry to the of the phosphorylation of the of for mass of mass of the the of method is it are for proteins are or the of the in of a K. 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The of mass spectrometry for the of protein phosphorylation sites has the past A analysis of methods in mass spectrometry is one method for of phosphorylation and the of have an of methods. the of a or is the of the of is of the of is in of mass spectrometry is in the for of the of phosphorylation. of mass trends the of the mass to phosphorylation site analysis in the of the and has an the in the of the and to a the mass the of the mass has in phosphorylation site mass such as the mass are the and of these has using for site Edman degradation has for in mass spectrometry has in phosphorylation site have the and of methods and the of methods in mass in phosphorylation site analysis that or phosphoproteins to using such as and the of the of is most various such as of protein are to and the of