Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

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Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome Matthias Mann, Shao-En Ong, Mads Grønborg, Hanno Steen, Ole N. Jensen and Akhilesh Pandey In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and peptides and discuss various options for their identification and quantitation with special emphasis on mass spectrometry-based techniques. Published online: 10 April 2002

Matthias Mann* Shao-En Ong Mads Grønborg Hanno Steen Ole N. Jensen Akhilesh Pandey* Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230, Denmark. *e-mails: [email protected] and [email protected]

Although prokaryotes mainly exhibit phosphorylation of histidine, glutamic acid and aspartic acid residues, phosphorylation of proteins on serine, threonine and tyrosine residues is recognized as a key mode of signal transduction and amplification in eukaryotic cells [1–3]. Here, we restrict our discussion to the study of phosphoproteins in eukaryotes – it is estimated that approximately one-third of all proteins in eukaryotic cells are phosphorylated at any given time (see Box 1) [4]. Phosphorylation of these proteins can modulate their activities by changing their 3D structure, as has been noted in several kinases. Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains specifically bind to phosphorylated tyrosine residues, whereas WW and Forkhead-associated (FHA) domains bind to phosphorylated serine and/or threonine residues [5,6]. A comprehensive analysis of protein phosphorylation (phosphoproteomics) involves identification of phosphoproteins and phosphopeptides, localization of the exact residues that are phosphorylated and quantitation of phosphorylation. However, analysis of phosphoproteins is not straightforward for five main reasons. First, the stoichiometry of phosphorylation is generally relatively low – only a small fraction of the available intracellular pool of a protein is phosphorylated at any given time as a result of a stimulus. Second, the phosphorylated sites on proteins might vary, implying that any given phosphoprotein is heterogeneous (i.e. it exists in several different phosphorylated forms). Third, many of the signaling molecules are present at low abundance within cells and, in these cases, enrichment is a prerequisite http://tibtech.trends.com

before analysis. Fourth, most analytical techniques used for studying protein phosphorylation have a limited dynamic range, which means that although major phosphorylation sites might be located easily, minor sites might be difficult to identify. Finally, phosphatases could dephosphorylate residues unless precautions are taken to inhibit their activity during preparation and purification steps of cell lysates. Several analytical techniques exist for the analysis of phosphorylation. Table 1 provides a comparison of mass spectrometry (MS)-based approaches to methods using Edman sequencing and 32P-phosphopeptide mapping for localization of phosphorylation sites. Readers are referred to several recent articles on various aspects of phosphoprotein and phosphopeptide analysis [3,7–12]. Enrichment strategies for phosphoprotein analysis

As mentioned, only a fraction of the proteins in a proteome are phosphorylated at any given time. Some of the most commonly used methods for enrichment of phosphoproteins or phosphopeptides when limiting amounts of the protein are available will now be discussed. These steps can be coupled to various analytical methods for detection and microcharacterization, as shown in Figure 1. Phosphospecific antibodies

Antibodies are routinely used to immunoprecipitate specific proteins. There are several commercially available antibodies that bind to phosphorylated tyrosine residues in a generic fashion. These antibodies can be used to immunoprecipitate, and therefore to enrich, tyrosine phosphorylated proteins from complex mixtures of proteins such as cell lysates. Although these antibodies have been relatively effective at enriching and identifying low-abundance tyrosine phosphorylated proteins [13], they are not very good at enriching for phosphopeptides [14], and thus other methods must be used. Currently, there are no antibodies that are suitable for enriching proteins that are phosphorylated on serine or threonine residues, and thus these proteins must be enriched using alternative methods.

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Box 1. Phosphorylation facts • Serine and threonine residues undergo phosphorylation more often than tyrosine residues.The phosphoamino acid content ratio (pSer:pThr:pTyr) of a vertebrate cell is 1800:200:1.There is a higher gain in signals involving tyrosine phosphorylation because it is less abundant and more tightly regulated. • One-third of all proteins in a eukaryotic cell are phosphorylated at any one time. • There are several commercially available antibodies that enrich for tyrosine phosphorylated proteins. Similar antibodies to enrich serine or threonine phosphorylated proteins are not currently available. • Phosphorylation is heterogeneous. Most phosphoproteins undergo phosphorylation on more than one residue – this does not mean that all the molecules of one protein are identically phosphorylated. • Kinases and phosphatases in the human genome. Approximately 2% of the human genome codes for kinases and phosphatases (there are ~500 kinases and 100 phosphatases in humans) [a]. Although the number of serine–threonine and dual specificity kinases is roughly four times that of tyrosine kinases, serine–threonine and dual specificity phosphatases almost equal the number of tyrosine phosphatases. • Phosphopeptides are generally difficult to analyze by mass spectrometry (MS) for several reasons.They are negatively charged whereas electrospray is generally performed in the positive mode. Being hydrophilic, they do not bind well to columns that are routinely used for purification of peptides before analysis. Phosphopeptides are not observed as intense peaks, especially in the presence of other nonphosphorylated peptides owing to ionic suppression. Finally, if the protease produces peptide fragments that are too small or too large, the peptides might not be observed in the mass spectrum at all. • Detection of phosphorylated serine, threonine or tyrosine residues using MS is not equally easy. Phosphoserine and phosphothreonine residues are labile (e.g. β-elimination, see Fig. 2a in main text), whereas phosphotyrosine residues are relatively more stable.This necessitates different MS strategies for their detection.

The use of miniaturized immobilized metal affinity chromatography (IMAC) columns for the enrichment of phosphopeptides exploits the high affinity of phosphate groups towards a metal-chelated stationary phase, especially Fe3+ and Ga3+. IMAC has been successfully used in off-line and on-line formats for the detection of phosphopeptides using MS [18–25]. Because it is based on the presence of negatively charged phosphate groups, IMAC generally enriches for phosphorylated serine, threonine and tyrosine residues. A major issue is that the specificity of this procedure is variable because of affinity for acidic groups (aspartic and glutamic acid) and to electron donors (e.g. histidine). In addition, multiply phosphorylated peptides are more enriched and the recovery of phosphopeptides appears to be largely dependent on the type of metal ion, column material and the elution procedure used. Recently, Ficarro et al. attained a much higher specificity using esterification of acidic residues before IMAC enrichment [26]. In this way they were able to sequence hundreds of phosphopeptides from total yeast protein extracts. With further refinement, this technique may offer the best hope for large-scale phosphorylation analysis. Chemical modification methods

Reference a Venter, J.C. et al. (2001) The sequence of the human genome. Science 291, 1304–1351

Phosphopeptide recovery by chromatographic methods

Peptides resulting from trypsin digestion of proteins are usually concentrated and desalted by purification over miniaturized reverse-phase C18 columns before analysis by electrospray MS. However, owing to the hydrophilic nature of phosphopeptides, significant losses can occur. Purification on a polymer-based reverse-phase perfusion chromatography resin (oligo R3), which was originally designed for purification of oligonucleotides [15,16], can usually enrich for phosphorylated and hydrophilic peptides. Alternatively, a porous graphitic carbon (PGC) column could be used to enrich for phosphorylated peptides [17].

Two methods have recently been reported that use chemical modification of the phosphate moiety as a strategy to enrich phosphopeptides from complex mixtures. The first method makes use of a β-elimination reaction that occurs when phosphoserine and phosphothreonine residues are exposed to strongly alkaline conditions [27,28] (Fig. 2a). The resulting dehydroalanine or dehydroaminobutyric acid residues can be detected using tandem mass spectrometry (MS/MS) or after chemical modification with ethanethiol [29,30]. Oda et al. used ethanedithiol (EDT) as a nucleophile, which provides a new reactive thiol group serving as a linker for attachment of a biotinylated affinity tag [27] (Fig. 2b). However, an undesired side effect involving side chains on cysteine and methionine residues can occur. To overcome this problem, the sample is first treated with performic acid, leading to

Table 1. Comparison of mass spectrometry-based techniques with other methods for phosphorylation analysis of peptides

Requirement for radioactivity Sensitivity Localization of sites

32

P-labeling and phosphopeptide mapping

Edman sequencing

Mass spectrometry

Large amounts required

May be used

Not required

Most sensitive Difficult without mutagenesis experiments Full coverage is difficult

Coverage High-throughput operation Purified protein required

Less sensitive (pmol) Possible. Tyrosine residues are problematic Full coverage is possible, provided sufficient material is available Not possible. Very labour intensive Difficult Yes Yes

Highly sensitive (fmol) Definitive localization is possible Full coverage is difficult. FTMS may provide full coverage Possible with automated LC-MS/MS setups No

Abbreviations: FTMS, Fourier transform mass spectrometry; LC-MS/MS, liquid chromatography tandem mass spectrometry.

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Mixture of phosphorylated and nonphosphorylated proteins Immunoprecipitation using phosphospecific antibodies

1D or 2D gel electrophoresis

In-solution digestion with trypsin

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Elute phosphopeptides from TLC plates

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Identify phosphopeptide

Chemical modification and biotin-tagging of phosphate containing residues

Affinity purification on avidin column

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Fig. 1. An overview of techniques for enrichment and analysis of phosphorylated proteins or peptides using mass spectrometry (MS)-based detection methods. MS/MS sequencing refers to tandem MS by any of the available instruments for this purpose.

oxidation of these residues, thereby inactivating them. An essentially identical strategy using a labeled version of EDT has been used for quantitation of phosphorylation and is described later [31]. The main disadvantages of this chemical modification method are that it is not applicable to tyrosine phosphorylation and that the yield from the β-elimination reactions tends to be sub-stoichiometric. Another problem is the low solubility of the thiol compound in water. However, because the reactions are performed in a single tube, this procedure is easier to perform than the method described below and losses resulting from multiple purification steps are minimized. The second method, developed by Zhou et al. [32], is applicable to phosphotyrosine-containing peptides in addition to those containing phosphoserine and phosphothreonine residues. The salient feature of this method is a transient carbodiimide [ethyl carbodiimide (EDC) was used in this case] catalyzed addition of cystamine to phosphate moieties, which allows purification of phosphopeptides on glass beads containing immobilized iodoacetyl groups (Fig. 2c). However, the amino groups of the peptides have to be protected first with tert-butyl oxycarbonyl (tBoc) chemistry and carboxyl groups by amidation to avoid http://tibtech.trends.com

unwanted reactions. Elution of phosphopeptides is performed by cleavage of phosphoroamidate bonds by trifluoroacetic acid, a step that also regenerates the amino groups. This approach requires several chemical reactions and purification steps before MS analysis, which could lead to substantial losses. As is generally the case with chemicalmodification-based approaches, both the above methods require large amounts of sample with the result that only abundant proteins are easily identified. Nevertheless, these approaches are promising and could be coupled to other fractionation steps to improve the overall recovery of low-abundance proteins. Detection of phosphorylation

Precursor ion scanning (MS/MS) – positive mode

Identify and sequence phosphopeptide directly

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Matrix-associated laser desorption ionisation (MALDI) has been successfully used for identification of proteins by ‘peptide mass fingerprinting’. In this technique, a list of peptide masses obtained by MALDI- time-of-flight MS (MALDI-TOF MS) from a proteolytic digest of a protein is compared with a theoretical digest of all the known proteins. However, analysis of phosphopeptides is not as straightforward as the identification of proteins using MALDI-TOF for several reasons. First, signals from phosphopeptides are usually suppressed because of the abundance of nonphosphorylated species as well as the weak ionization of phosphopeptides in positive mode. Second, the presence of isobaric peptides in the sample can complicate analysis. Third, peptide mass fingerprinting does not yield direct sequence information, thus identification of the exact phosphorylated residue is not possible. Finally, purified protein or peptides are required for analysis of phosphorylation by this technique, which requires prior purification steps by one or two-dimensional electrophoretic gels or by high performance liquid chromatography (HPLC). In an attempt to overcome some of these drawbacks, MALDI-TOF MS has been used in combination with phosphatase treatment to specifically identify phosphopeptides [21,33,34]. The aim is to identify phosphopeptides based on a characteristic mass shift owing to loss of phosphate (80 Da or multiples) after treatment with phosphatase. However, the usual problems associated with analysis of peptide mixtures in MALDI preclude complete sequence coverage of the protein. It is sometimes possible to differentiate between serine or threonine and tyrosine phosphorylation on phosphopeptides using MALDI-TOF. In the positive ion mode, the tendency for serine or threonine phosphopeptides to show a predominant neutral loss of 98 Da (owing to H3PO4 loss) as compared with a loss of 80 Da (owing to HPO3 loss) can be used to differentiate them from tyrosine phosphopeptides, which generally show only a loss of 80 Da [35]. MALDI-TOF MS can be valuable if performed on peptide mixtures that are first purified on IMAC columns to enrich for phosphorylated peptides.

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Fig. 2. Enrichment strategies using chemical modifications. (a) β-Elimination reaction: under strongly alkaline conditions, phosphoserine and phosphothreonine residues undergo an elimination reaction whereby phosphoric acid is lost and an α,β unsaturated bond is formed.The end products are dehydroalanine and dehydroaminobutyric acid, respectively, as shown. A similar reaction occurs in the gas phase when collision-induced dissociation (CID) experiments are performed. (b) Chemical modification based on β-elimination: samples containing phosphoproteins are first treated with a strong base, leading to β-elimination reaction in the case of phosphoserine (pictured here) and phosphothreonine residues. A reactive species containing an α,β unsaturated bond is formed.This serves as a Michael acceptor for the nucleophile (in this case, ethanedithiol or an isotopic variant may be substituted for quantitation purposes).The biotinylated reagent reacts with sulfhydryl (-SH) groups at acidic to neutral pH. Biotinylated phosphoprotein is now tagged for enrichment on avidin columns in later steps. (c) Chemical modification based on carbodiimide condensation reaction.The amino termini of peptides are first protected with tert-butyl oxycarbonyl (tBoc) chemistry. Following this, a condensation reaction occurs between the carboxyl groups as well as the phosphate moiety in the presence of excess amine (ethanolamine) in a reaction catalyzed by N, N ′-dimethylaminopropyl ethyl cabodiimide HCl (EDC).The condensation reaction results in the formation of an amide bond and a phosphoamidate bond from carboxyl and phosphate bonds, respectively.The phosphate group is regenerated by rapid hydrolysis with acid and the sample is desalted on reverse phase material (this intermediate step is not shown here). A second condensation reaction (also catalyzed by EDC) is performed next with excess cystamine.The sample is reduced with dithiothreitol (DTT), converting the disulfide bond of cystamine to a sulfhydryl group and thereby tagging the phosphate moiety.The sample is again desalted using reverse phase material. The tagged peptides are captured on glass beads containing bound iodoacetyl groups that will react with sulfhydryl groups. The recovery of phosphopeptides is performed by strong acid hydrolysis that cleaves both the phosphoamidate bond and the tBoc protective group, thus regenerating the phosphate moiety and the N-terminus, respectively.

This method enables the visualization of peptides that are otherwise not observed in the MS spectrum because of suppression effects. Recently, MALDI MS in the negative ion mode has been used for identification of phosphopeptides from simple mixtures. In these preliminary studies, somewhat more intense signals were found for phosphopeptides in the negative as compared with the positive ion mode [36]. An additional experiment, based on post source decay (PSD) is sometimes used to generate sequence information that confirms the site of phosphorylation during analysis by MALDI-TOF [35,37,38]. However, the difficulty in obtaining good quality PSD spectra limits the use of this technique, but could be overcome by newer instruments. Coupling of MALDI to tandem mass spectrometers such as quadrupole-TOF (MALDI q-TOF), http://tibtech.trends.com

TOF-TOF (MALDI-TOF-TOF) or ion trap (MALDI-ion trap) would not only provide rapid identification of phosphopeptides but also subsequent localization of the phosphorylation sites based on MS/MS sequencing [39–44]. It is to be noted that some of the drawbacks of MALDI are not overcome by the use of these instruments. Therefore, enrichment of phosphopeptides would still be of great value. These advances in instrumentation are relatively new and, therefore, their performance remains to be determined. Precursor ion scanning by tandem MS

On fragmentation by collision-induced dissociation (CID) in a tandem mass spectrometer, phosphopeptides not only produce sequence-specific fragments but also fragments that are specific for phosphate groups. These phosphate-specific fragment ions serve as characteristic ‘reporter ions’ for phosphorylated peptides in precursor-ion scanning experiments by MS/MS. Peptides carrying a phosphate group can therefore be easily identified by precursor-ion scanning because of the loss of phosphate (PO3−) under alkaline conditions [45,46]. A triple quadrupole mass spectrometer operating in negative ion mode is generally used for this purpose. In this method, detection of the specific reporter ion identifies the corresponding precursor phosphopeptide ion by its mass to charge (m/z) value. Subsequent sequencing of the corresponding phosphopeptide requires a change in polarity and rebuffering of the sample, which implies that this system is not readily amenable to liquid chromatography (LC)-MS-based approaches. Despite these shortcomings, the method is a powerful tool because of its high selectivity and sensitivity and its applicability for serine, threonine and tyrosine phosphorylated residues. A precursor-ion scanning method that can be performed in the positive mode has recently been developed for the specific detection of

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phosphotyrosine-containing peptides [47,48]. This method is based on the ability to selectively detect the immonium ions of phosphotyrosine residues that have an m/z value of 216.043. Immonium ions are generated by double cleavage of the peptide backbone. Using newer high-resolution instruments, such as those operating on the q-TOF principle (with a Q2-pulsing function) [49], these immonium ions can be easily distinguished from other peptide fragment ions. Phosphopeptides that are barely detected in the original MS scan can be easily identified using this approach. Once the phosphotyrosine-containing peptides are located, they can be sequenced in the product ion MS/MS mode without any need for switching polarity of the ion source. This scanning method is sensitive and tyrosine phosphorylation sites from subpicomole amounts of gel-separated proteins have been successfully identified. Unfortunately, owing to the lability of phosphoserine and phosphothreonine residues, this method cannot be applied for identification of phosphorylation events involving these amino acids and is not yet suitable for LC-MS/MS. Scanning for neutral loss of 98 (H3P04) or 80 (HPO3)

When peptides containing phosphoserine or phosphothreonine residues are subjected to CID, they commonly undergo a gas-phase β-elimination reaction, resulting in a neutral loss of phosphoric acid (−98 Da) or are dephosphorylated (−80 Da). Phosphotyrosines, however, are generally more resistant to this loss. Because m/z values and not absolute masses are measured in a mass spectrometer, doubly and triply charged peptide ions show an apparent loss of 49 and 32.66 Th (Thompson) in the mass spectrum, respectively. Ideally, precursor ion experiments can be performed on a triple quadruple mass spectrometer with an offset to detect phosphopeptide species that undergo such a loss [50–52]. In the MS/MS spectrum, a spacing of 69 Da (owing to dehydroalanine) or 83 Da (owing to dehydroaminobutyric acid) indicates the exact location of phosphorylated serine and threonine residues, respectively. The drawbacks of this method are the incidence of false-positive signals as well as the fact that the charge state of the phosphopeptide has to be known in advance. It is also possible to use q-TOF mass spectrometers for such experiments in an automated data-dependent acquisition mode. LC-MS/MS

Separation of tryptic peptides using LC is an excellent way to decrease the complexity of the sample. In this method, peptides are first loaded onto a nanocolumn (usually 75 µm internal diameter) containing reverse phase C18 material and then eluted by using a gradient directly into a tandem mass spectrometer, generally an ion-trap instrument but also increasingly a q-TOF instrument, which provides a http://tibtech.trends.com

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higher resolution. The peptides are eluted at a slow flow rate, typically 100–200 nl per minute, and the elution of a peptide generally occurs in a peak lasting ~10–30 s. Hundreds to thousands of peptides can therefore be separated and analyzed using this method [53]. In a variation of this technique, a 2D chromatographic separation, first on a strong cation exchange and then on a C18 column, is performed [54]. As the peptides elute off the column into the mass spectrometer, a mass spectrum in the survey scan mode is obtained. In the data-dependent acquisition mode, the instrument can be set to automatically fragment and collect MS/MS data on any number of peaks observed in the MS spectrum based on their intensity, m/z value or charge state. If an ion-trap mass spectrometer is used, the sensitivity can be as high as 1–5 fmol in the MS mode; however, when operated in the MS/MS mode, the sensitivity increases 10–20-fold. Coupling of nanoLC systems to a mass spectrometer is valuable because separation of peptides by the upfront LC step decreases the ion suppression effect observed in the case of phosphopeptides. This method has been successfully used for analysis of phosphorylation sites in several cases [55–57]. In one of the variations of this method, a simple mixture containing one or two proteins can be digested by two different proteases followed by LC-MS/MS. A search algorithm can be used to assign the phosphorylation site based on the fragmentation spectra provided only a small database is searched (Steven P. Gygi, pers. commun.). Electron capture dissociation by Fourier transform MS

Electron capture dissociation (ECD) combined with Fourier transform ion cyclotron resonance (FTICR) MS has emerged as a powerful method for the sequencing of proteins and peptides as well as for the study of post-translational modifications [58]. Recently, it has also been successfully applied for the exact localization of phosphorylated residues in peptides [59,60]. ECD induces more extensive fragmentation of the peptide backbone than CID, providing greater sequence coverage. An advantage of ECD is its applicability for identification of phosphorylated residues. In contrast to conventional CID and PSD, no loss of phosphoric acid, phosphate or water from the parent peptide or the fragments is seen when ECD-based sequencing of phosphopeptides is performed. This allows direct assignment of phosphorylation sites. Owing to the extremely high resolution of FTMS (>10 times higher than other mass spectrometers), large peptides and proteins that are not amenable to conventional MS can also be studied [61,62]. This means that, unlike other MS methods in which only a tryptic peptide is analyzed, studying the whole protein by FTICR will provide a more comprehensive picture of the phosphorylation status of the protein. The most significant limitation of this

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Phosphoamino acid analysis and stable isotopic labeling + Sample 1

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Fig. 3. Quantitation of phosphopeptides using mass spectrometry (MS).This schematic diagram shows enrichment of phosphopeptides from two states labeled with isotopically distinct biotinylated mass tags (colored blue and orange). After labeling, the samples are mixed and purified over an avidin column.The unbound peptides are removed by washing followed by elution of tagged peptides and analysis by MS. The mass spectrum below shows pairs of peaks for formerly phosphorylated peptides owing to the mass difference introduced by biotin tags of two different masses.The relative amounts of phosphopeptides in the two states can be derived from the relative intensities of the two peaks. The illustration shows one peptide (shown in black) that is differentially phosphorylated between the two states, whereas the other peptide (shown in orange) is phosphorylated to the same extent in both states.

technique is the requirement for relatively pure samples for ECD experiments and the availability of expensive instrumentation and trained personnel. Studying cellular dynamics: quantitation of protein phosphorylation

There are several reasons why quantitation of phosphorylation is particularly important. A given protein might be in more than one signaling pathway (as is usually the case) with different stimuli inducing overlapping patterns of phosphorylation. That means a given site might not be phosphorylated at all, phosphorylated in a minority of molecules or, in an extreme case, on all the molecules of that protein. When a population of molecules from unsynchronized cells is analyzed, this situation corresponds to detection of unphosphorylated, weakly phosphorylated or highly phosphorylated peptides containing the residue. Similarly, the ratio of phosphorylation of a protein on multiple residues might be crucial for its function. The techniques that have traditionally been used for quantitation of phosphorylation are phosphoamino acid analysis and Edman degradation, although MS-based techniques are rapidly evolving. http://tibtech.trends.com

A 32P-labeled protein can be hydrolyzed into its constituent amino acids and resolved using electrophoresis [63]. This provides quantitation of an increase or decrease in phosphorylation of the labeled protein under different conditions. This is not a high-throughput procedure and requires enrichment of the protein of interest. Another drawback of this method is that it measures the incorporation and not the steady-state level of phosphorylation. Thus, it is dependent on the rate of turnover of proteins as well as the kinetics of the relevant kinases and phosphatases in the cell, leading to potentially biased results in terms of quantitation. Labeling of proteins with a stable isotope such as 15N allows the measurement of the relative amounts of proteins by comparing ion intensities of corresponding peptides when unlabeled and labeled are mixed and analyzed. This method can also be used for quantitation of phosphorylated peptides because the relative intensities of 14N and 15N peaks for the phosphorylated and nonphosphorylated versions of a given peptide provide a measure of the extent of phosphorylation [64]. This method only provides a quantitation at the level of the phosphopeptides; sequencing by MS/MS is still required to determine the actual positions of phosphorylated residues within the peptide. Also, this method can only be used in cases in which the stable isotope can be incorporated into proteins (i.e. in cell culture). Another limitation of this method is that although it is useful for quantitation, it does not enrich for phosphorylated proteins and therefore suppression of signals from phosphopeptides is still observed. Therefore, it has to be coupled to other enrichment methods to achieve higher throughput. Its major advantage is that it is simple to perform. The recent use of isotope coded affinity tags (ICAT) as mass tags for quantitation of protein levels has been further extended to quantitation of phosphorylation [65]. Use of a β-elimination reaction to introduce biotin-containing affinity tags with two different masses, termed PhIAT (phosphoprotein isotope coded affinity tags), is one way to quantitate peptides containing phosphorylated serine and threonine residues [31] (Fig. 3). In an alternative approach, Weckwerth et al. replaced the phosphate moiety in phosphoserine- and phosphothreoninecontaining peptides by β-elimination, followed by Michael addition of ethanethiol or its fully deuterated version [66]. No enrichment was performed in this case after labeling of the two samples. Although it is enticing to think that such quantitation methods will become the mainstay of phosphorylation analysis, it remains to be seen if these methods can be used for global approaches because chemical reaction yields are low and phosphorylated species constitute only a minority of the total protein. However, we feel that such techniques have great potential if they are

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applied to specific proteins or a subset of proteins, especially when larger amounts of proteins are available. Elemental MS Acknowledgements We thank Jens Andersen and other members of the Protein Interaction Laboratory for useful discussions. We also thank Peter Højrup for helpful comments. Work at the Center for Experimental Bioinformatics is supported by a generous grant from the Danish National Research Foundation. Akhilesh Pandey is supported by the HowardTemin Award (National Cancer Institute, CA 75447) and by a travel award fromThe Plasmid Foundation, Denmark.

Isotopic labeling is not required if LC-MS is performed in conjunction with an elemental MS method such as inductively coupled plasma (ICP) ionization. As the ionization of peptides is not affected by suppression effects or varying ionization responses, absolute quantitation can be obtained with sensitivity in the subpicomole range [67]. However, the requirement for special instrumentation limits the use of this technique. Outlook

As is obvious from the range of techniques described above, there is no single method that supersedes all others for the identification and localization of phosphorylation sites. Important parameters in deciding the most appropriate analytical method are

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quantity of the protein available, whether serine or threonine or tyrosine residues are phosphorylated, whether purified protein is available for analysis and finally if a global analysis is desired. Regardless of the method used, enrichment of phosphorylated protein or peptide improves the likelihood of success. Although MS is certainly the technique of choice for phosphopeptide detection and sequencing, its limitations must always be borne in mind. This means that comprehensive analysis might still require the use of traditional mutagenesis or 32P-labeling methods. Nevertheless, techniques for identification and localization of phosphorylation sites in proteins are more accessible today, mainly because of the advances in sample preparation techniques as well as instrumentation. Better enrichment strategies and quantitation of phosphorylation are the main challenges in this rapidly evolving field. With continued development, it should be possible to perform a global analysis of protein phosphorylation in the near future.

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