591 Phosphorylation on serine, threonine and tyrosine residues is an extremely important modulator of protein function. Therefore, there is a great need for methods capable of accurately elucidating sites of phosphorylation. Although full characterization of phosphoproteins remains a formidable analytical challenge, mass spectrometry has emerged as an increasingly viable tool for this task. This review summarizes the methodologies currently available for the analysis of phosphoproteins by mass spectrometry, including enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopeptides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags. Despite the variety of powerful analytical methods that are now available, complete characterization of the phosphorylation state of a protein isolated in small quantities from a biological sample remains far from routine. Addresses The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA *e-mail: mclachd@mail.rockefeller.edu † e-mail: chait@mail.rockefeller.edu Current Opinion in Chemical Biology 2001, 5:591–602 1367-5931/01/$ —see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations CE capillary electrophoresis CID collision-induced dissociation EGF epidermal growth factor ESI electrospray ionization HPLC high-performance liquid chromatography ICP inductively coupled plasma IMAC immobilized metal affinity chromatography MALDI matrix-assisted laser desorption/ionization MS mass spectrometry Q1 first quadrupole Q2 second quadrupole Q3 third quadrupole Qq quadrupole–quadrupole TOF time-of-flight Introduction Organisms use reversible phosphorylation of proteins to control many cellular processes including signal transduc- tion, gene expression, the cell cycle, cytoskeletal regulation and apoptosis [1,2]. Although phosphorylation is observed on a variety of amino acid residues, by far the most common and important sites of phosphorylation in eukaryotes occur on serine, threonine and tyrosine residues. Because of the central role of phosphorylation in the regulation of life, much effort has been focused on the development of methods for characterizing protein phosphorylation. Traditional methods for analyzing O-phosphorylation sites [3–5] involve incorporation of 32 P into cellular proteins via treatment with radiolabeled ATP. The radioactive proteins can be detected during subsequent fractionation procedures (e.g. two-dimensional gel electrophoresis or high-perfor- mance liquid chromatography [HPLC]). Proteins thus identified can be subjected to complete hydrolysis and the phosphoamino acid content determined. The site(s) of phosphorylation can be determined by proteolytic digestion of the radiolabeled protein, separation and detection of phosphorylated peptides (e.g. by two-dimensional peptide mapping), followed by peptide sequencing by Edman degradation. These techniques can be tedious, require significant quantities of the phosphorylated protein and involve the use of considerable amounts of radioactivity. In recent years, mass spectrometry (MS) has become an increasingly viable alternative to more traditional methods of phosphorylation analysis [6–8]. In this review, we out- line the various mass spectrometric techniques that can be used to characterize sites of O-phosphorylation (Box 1). In particular, we concentrate on methods that do not involve the use of 32 P, although MS can certainly be used in conjunction with the classical methods of phosphoprotein and phosphopeptide analysis mentioned above (e.g. [9]). MS can accurately provide the molecular mass of the intact phosphorylated protein. Such measurements, used in conjunction with calculation of the molecular mass of the unmodified protein [10] and/or treatment with phos- phatase [11–13] allow determination of the average number of attached phosphate groups. Provided that the MS resolution is sufficiently high, it is also feasible to determine the distribution of the number of attached phosphates [14]. For more detailed analysis of the sites of phosphate attachment and stoichiometry, it is necessary to examine peptide fragments of the phosphoprotein of inter- est. Such fragments are usually generated by digestion of the phosphoprotein with site-specific proteases such as trypsin. Most of the work described here relates to MS measurements of phosphopeptides. Sample preparation Analysis of phosphopeptides presents formidable challenges to the mass spectrometrist. Ideally, every phosphorylated component of the protein should be detected. Unfortunately, MS analysis of proteolytic digests of proteins rarely provides 100% coverage of the protein sequence, and regions of inter- est are easily missed. In addition, negatively charged modifications can hinder proteolytic digestion by trypsin, the protease of choice in many applications. Phosphorylation is often sub-stoichiometric, such that the phosphopeptide is Analysis of phosphorylated proteins and peptides by mass spectrometry Derek T McLachlin* and Brian T Chait †