Repair of pectus excavatum using a dacron vascular graft strut

Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry Ole Nørregaard Jensen Post-translational modifications generate tremendous diversity, complexity and heterogeneity of gene products, and their determination is one of the main challenges in proteomics research. Recent developments in mass spectrometry based approaches for systematic, qualitative and quantitative determination of modified proteins promise to bring new insights on the dynamics and spatio-temporal control of protein activities by post-translational modifications, and reveal their roles in biological processes and pathogenic conditions. Combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies and mass spectrometry are particularly attractive for systematic investigation of post-translationally modified proteins in proteomics. Addresses Protein Research Group, Department of Biochemistry & Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark e-mail: [email protected]

Current Opinion in Chemical Biology 2004, 8:33–41 This review comes from a themed issue on Proteomics and genomics Edited by Michael Snyder and John Yates III 1367-5931/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2003.12.009

Abbreviations 2DE ECD FT-ICR GPI IMAC LC MALDI MS (MS/MS) PTM TOF

two-dimensional gel electrophoresis electron capture dissociation Fourier transform ion cyclotron resonance glycosylphosphatidylinositol immobilized metal ion affinity chromatography liquid chromatography matrix-assisted laser desorption/ionization (tandem) mass spectrometry post-translational modification time-of-flight

Introduction Living cells rely on a multitude of interrelated dynamic molecular processes that govern cellular growth, reproduction and survival. These intricate biological processes are mediated mainly by protein molecules, which interact in a highly dynamic fashion with each other and with metabolites, phospholipids, carbohydrates and nucleic acids. Protein activity is not only controlled by the rates of protein biosynthesis and protein degradation but also by specific and selective covalent processing (i.e. posttranslational modification, PTM), which modulates molewww.sciencedirect.com

cular interactions, protein localization and stability. Failure to control complex molecular processes is detrimental or fatal for the survival of the cell and it is no surprise that a range of post-translationally modified proteins and their substrates are implicated in human diseases and ailments, including cancers, diabetes, the dysmetabolic syndrome, and numerous neurological disorders. PTMs are chemical alterations to protein structure, typically catalyzed by exceedingly substrate-specific enzymes, which themselves are under strict control by PTMs. More than 300 different types of PTMs are currently known and new ones are regularly discovered. Thus, a gene product can be present in several different modified forms, including various splice variants; multiple phosphorylated, glycosylated and acylated forms (Figure 1). Combinations of different, sub-stoichiometric PTMs give rise to heterogeneity of the protein population. This, in turn, makes it a highly demanding task to achieve complete characterization and accurate quantitation of posttranslationally modified proteins. PTM and processing of a protein leads to a mass increment or a mass deficit relative to the molecular weight calculated from the naked amino acid sequence of the protein. Phosphorylation of a serine residue leads to a mass increment of 80 Da, thereby increasing the nominal molecular mass of this residue from 87 Da to 167 Da. This mass increment can in principle be detected by accurate mass determination by mass spectrometry of the intact protein, an approach often used in the biotechnology industry to monitor recombinant proteins. Traditionally, the modified regions of proteins are further characterized by peptide mass mapping subsequent to enzymatic digestion of the protein. In this way, the molecular masses of individual peptides are examined by comparison to the corresponding calculated (expected) molecular masses. Finally, the modified amino acid residues are determined by sequencing by tandem mass spectrometry (MS/MS). Although this approach for determination of PTMs sounds very simple, it is a non-trivial task to characterize PTMs by MS. This is mainly because of the complexity of the post-translationally modified protein sample and the physicochemical characteristics and size of modified peptides, which in turn lowers their detection efficiency by MS. In this review, a range of recently reported MS-based approaches for determination of PTMs are discussed. Several of these approaches for proteome-wide PTM analysis take advantage of affinity-based enrichment Current Opinion in Chemical Biology 2004, 8:33–41

34 Proteomics and genomics

Figure 1

show great promise for the detection, characterization and quantitation of PTMs [1–3] (Table 1).

Genome

Transcriptome

Proteome

Co/post-translational processing

mRNAD

Protein1 Protein2 Protein3 Protein4 Protein5 Protein6

mRNAE

....

Alternative splicing mRNAA mRNAB Genex

mRNAC

2DE and MS for characterization of post-translationally modified proteins Two-dimensional gel electrophoresis (2DE) is a powerful method for the separation and isolation of proteins [4]. Based on orthogonal separation of proteins by isoelectric point and molecular weight, it can often resolve similar but differentially modified forms of a given protein. Many 2DE-based methods for studying PTMs rely on selective and specific probes for the detection of intact modified proteins in the gel or after blotting onto membranes (Table 1). In addition, numerous approaches for the enrichment of modified proteins before electrophoresis and MS analysis have emerged.

Proteini Current Opinion in Chemical Biology

From genome to proteome: a major increase in complexity and dynamics. The diversity of gene products originating from a single gene is mainly due to alternative splicing of transcripts and co- and posttranslational modification of proteins. The human genome is predicted to contain on the order of 30 000 open reading frames, each of which, on average, may produce five or six different mRNA species. Each of these mRNA species are in turn translated into proteins that are processed in various ways, generating on the order of 8–10 different modified forms of each polypeptide chain. Thus, the human genome may potentially produce on the order of (30 000  6  10) 1.8 million different protein species.

methods or apply ‘capture and release’ techniques to isolate subsets of modified proteins. MS-based techniques optimized for separation and sequencing of peptides are complementary to electrophoretic methods for separation of intact proteins. Combinations of these technologies

Different phosphorylated forms of a protein can be visualized by 2DE (Figure 2) [5]. The ‘pearls-on-string’ pattern is a telltale indication of protein phosphorylation, although the introduction of charge heterogeneity by deamidation of Asn or Gln residues to form carboxylic acids can also generate such a pattern. Parallel phosphatase treatment of an identical sample generates a differential 2DE readout for the presence or absence of phosphoprotein spots, thereby improving the reliability of phosphoprotein assignments [6]. Radiolabeling of proteins by 32-P incorporation is a useful method for specific detection of phosphoproteins in 2DE gels, as recently shown for plant mitochondrial phosphoproteins (Figure 2). Phosphopeptide and phosphoamino acid assignments of several phosphoproteins from the 2DE gel were subsequently achieved by matrix-assisted laser desorption/ ionization time-of-flight (MALDI TOF) MS, nanoelectrospray MS/MS and liquid chromatography (LC)-MS/ MS analysis [5]. Incorporation of 32-P in proteins can also

Figure 2

(a)

Coomassie-staining

5 Molecular mass (kDa)

SDS-PAGE

IEF

pl

(b) 8 5

Phosphorimage pl

8

45

30 (c)

(d)

45

30

Phosphoprotein analysis by 2DE. Mitochondrial proteins from potato tubers were separated by 2DE and visualized by (a,c) Coomassie staining or (b,d) by 32-P incorporation and phosphorimaging. The total mitochondrial fraction (a,b) and the mitochondrial matrix fraction (c,d) were analyzed. The indicated protein spots correspond to pyruvate dehydrogenase and formate dehydrogenase, which are comigrating to generate partially overlapping protein spots. Both proteins were assigned as phosphoproteins based on 32-P labeling. MS was subsequently used to determine phosphorylation sites in both proteins. Adapted from [5] with permission. Current Opinion in Chemical Biology 2004, 8:33–41

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Modification-specific proteomics Jensen 35

Table 1 Strategies, techniques and reagents used in modification specific proteomics. Detection Metabolic labeling Western blotting Staining PTM-specific probes Affinity enrichment Epitope binders Chelators Affinity tagging Chemical conversion/addition

Method

Phosphoproteins

Glycoproteins

Other PTMs

Radioactive isotope incorporation Antibodies (Ab) Fluorescent probes Enzymes

32

3H (GlcNAc)

3H (lipids)

a-pY Ab, a-pS/pT Ab Pro-Q Diamond Phosphatase, kinase

Conjugated lectins Pro-Q Emerald Glycosidase

a-nitroTyr Ab

Antibodies Lectins Immobilized ions/ligands

a-pY, a-pS/pT Fe(III)-IMAC, Ga(III)-IMAC

ConA, WGA Boronic acid

Beta elimination/Michael addition Phosphoramidate chemistry Reduction and alkylation

Biotinylation (pS/pT)

Alkylation (O-GlcNAc)

P (pY, pS, pT, others)

a-nitroTyr Ab Ni(III), His6-tag (Ubiquitin)

Covalent capture (pY/pS/pT) Biotinylation (nitroTyr)

Mass tagging Chemical conversion/addition Stable isotope mass tagging

Beta elimination/Michael addition Enzymatic incorporation of isotope via hydrolysis

DMass signature (pS/pT)

Mass spectrometry Mass determination Sequencing Selective detection

MS MS/MS product ion scan MS/MS precursor ion scan

DMass (80) DMass (80, 98) 216 (pY), 79 (pS/pT/pY)

Quantitation

Lipases (GPI-anchors)

DMass (O-GlcNAc) Glycosidase,

MS/MS neutral loss scan 98 (H3PO4) In vivo/in vitro stable isotope Metabolic/chemical labeling labeling of proteins or peptides

18

O-water

DMass (162, 203, 291) DMass (162, 203, 291) 163 (Hex), 204 (HexNAc)

Protease, 18 O-water (C-term. processing)

Metabolic/chemical labeling

203 (HexNac) Metabolic/chemical labeling

Modification-specific proteomics takes advantage of selective and specific reagents for sensitive detection, affinity-purification and mass spectrometric analysis of post-translationally modified proteins. Refer to the main text for details and references.

facilitate phosphoamino acid determination by chemical methods, such as Edman degradation of peptides, when combined with computational sequence analysis [7,8]. Several novel fluorescent dyes have been developed for modification-specific staining of glycoproteins and phosphoproteins in electrophoretic gels to enable multiplex analysis of post-translationally modified proteins [9]. Western blotting using anti-phosphoamino acid antibodies for detection of phosphoproteins, conjugated lectins for detection of glycoproteins and anti-nitrotyrosine antibodies for detection of nitrated proteins are generally used for modification-specific visualization of proteins in 2DE gels. In such cases, it is important to keep in mind that subsequent MS-based characterization of proteins seen in 2DE gels by using modification-specific probes can be obscured by the presence of abundant, unmodified co-migrating proteins. It is therefore advantageous to reduce the complexity of protein samples that are to be analyzed by 2DE (e.g. by prior purification or enrichment of modified proteins).

Affinity-enrichment methods Affinity-based enrichment of post-translationally modified proteins and peptides serves to increase the relative www.sciencedirect.com

abundance of a select class of modified polypeptide species in a sample. It is applied at the intact protein level or at the peptide level (i.e. after enzymatic or chemical degradation of proteins) (Table 1). Anti-phosphotyrosine antibodies are extensively used for immunoprecipitation of intact phosphoproteins in MSdriven proteomic strategies to study cell signaling [10,11]. Anti-pSer/pThr antibodies also facilitate enrichment of phosphoproteins, thereby revealing novel kinase substrates [12,13]. The development and availability of a broader range of specific anti-phosphoamino acid antibodies for immunoprecipitation should permit detailed analysis of phosphoproteome dynamics in the future. Immobilized metal ion affinity chromatography (IMAC) takes advantage of the affinity of chelated Fe(III) or Ga(III) ions towards the phosphate group of phosphopeptides. IMAC is widely used for isolating phosphopeptides from relatively simple peptide mixtures before MS [14]. In addition, the technique has emerged in phosphoproteomics to enrich phosphopeptides from crude peptide mixtures generated by enzymatic digestion of yeast cell lysates [15], after affinity-purification of human Current Opinion in Chemical Biology 2004, 8:33–41

36 Proteomics and genomics

phosphotyrosine-containing proteins [11] or after isolation of plant membrane proteins [16]. The IMAC method has been refined by using either O-methylesterification of carboxylic groups to reduce non-specific binding [15] or by using strong anion exchange (SAX) chromatography to reduce sample complexity before IMAC [16]. These methods facilitate the recovery, sequencing and assignment of hundreds of phosphopeptides from complex protein samples. Lectins are carbohydrate binding proteins that recognize specific carbohydrate structures, and are used to enrich for glycoproteins and glycopeptides [17,18]. Lectin-based affinity enrichment of glycopeptides in combination with glycosidase-catalyzed 18-O stable isotope labeling and MS/MS permits isolation, detection and sequencing of Nglycosylated peptides [19]. Hydrolytic incorporation of 18-O at the glycosylation site facilitates MS- and MS/MSbased assignment of glycopeptides and exact determination of N-glycosylation sites. This method revealed 400 N-glycosylation sites in 250 glycoproteins in a Caenorhabditis elegans protein extract. Ubiquitination, which destines proteins for destruction, was investigated on a proteome-wide scale by combining genetic, biochemical and MS techniques [20]. Expression of His6-tagged ubiquitin instead of the native polypeptide enabled affinity isolation of the ubiquitinconjugated pool of proteins from a yeast cell lysate. MS identified hundreds of ubiquitinated protein candidates and also revealed more than 70 ubiquitinylated peptides. These recent examples clearly demonstrate the feasibility and promise of using affinity-enrichment methods in modification-specific proteomics. These strategies are further extended by taking advantage of organic chemistry to incorporate modification-specific probes into proteins and peptides, as outlined below.

Affinity-tagging: chemical techniques in modification-specific proteomics Chemical approaches for conversion of post-translationally modified amino acid residues into more tractable species are increasingly used. Introduction of affinity tags by b-elimination of phosphoric acid from pSer and pThr followed by Michael addition of affinity-groups, such as biotin, enable enrichment of phosphopeptides and phosphoproteins [21,22] (Table 1). This approach has also been used to introduce phosphorylation-specific ‘mass tag signatures’ in the form of acyl groups [23] for detection by MALDI MS, and for introduction of reporter groups that generate diagnostic fragment ion signals for use in MS/MS precursor ion scanning [24]. A novel chemoenzymatic approach designed for phosphoprotein analysis by MS uses b-elimination followed by addition of cysteamine to convert phosphoserine and phosphothreonine residues into aminoethylcysteine residues, which are lysine analogues. Thus, phosphoamino acid specific cleaCurrent Opinion in Chemical Biology 2004, 8:33–41

vage sites for trypsin and endoproteinase Lys-C are generated, leading to facile assignment of phosphorylation sites by MS [25] (see also Update). Common to all techniques that utilize the b-elimination reaction for phosphoprotein analysis is that they are only applicable to phosphoserine and phosphothreonine residues, but not to phosphotyrosine residues. However, a technique based on phosphoramidate chemistry enables the conversion and capture of all three types of phosphorylated residues [26]. O-glycosylated amino acid residues also undergo b-elimination and this feature was explored in a proteomic analysis aimed at assigning O-GlcNAc modified proteins [27]. The importance of discriminating O-phosphorylation and O-glycosylation of proteins was emphasized [27]. A recently reported method for affinity tagging of nitrotyrosine residues takes advantage of strong reducing agents to convert nitrotyrosine into aminotyrosine followed by biotinylation of this residue to facilitate affinity enrichment [28]. This method complements proteomic methods based on 2DE and western blotting for detection and identification of nitrated proteins [29]. In contrast to the above method, a glycoproteomics strategy used oxidation to activate carbohydrates in glycoproteins for their immobilization onto a hydrazide-activated resin. Subsequent proteolysis, derivatization and MS facilitated relative quantitation and identification of glycoproteins and the assignment of N-glycosylation sites (Figure 3) [30]. The chemical approaches for introducing affinity tags or mass tags usually require multiple processing and desalting steps, which often reduces the overall sensitivity in proteomics studies. Immobilization or covalent capture of the target molecules seems a most promising approach, as it allows extensive washing of the targeted sample without significant loss in sensitivity.

Characterization of post-translationally modified membrane proteins Membrane proteins are associated with or embedded in the lipid bilayer of cellular membranes, such as the plasma membrane, the endoplasmic reticulum and the nuclear membrane. Eukaryote plasma membrane proteins are often heavily post-translationally modified in the extracellular domains (e.g. N-glycosylation) and in the intracellular domains (e.g. phosphorylation or acylation). Because of their extensive hydrophobic regions, integral membrane proteins present a special challenge to proteome analysis because they are insoluble under standard aqueous conditions. Several promising MS- based proteomic strategies for the identification and characterization of membrane proteins have appeared. In one approach, the isolated membrane fraction is treated with www.sciencedirect.com

Modification-specific proteomics Jensen 37

Figure 3

(c)

(a)

Phosphorylated membrane proteins

Invert vesicles

'Shave'

Enrich phosphopeptides

Brij-58

Trypsin

Fe(III)-IMAC

Plasma membrane preparation (vesicles)

PI-PLC

LC

MS/MS

GPI-anchored membrane proteins

(d) ’Shave’ vesicles

Separate Sequence phosphopeptides

Extract proteins

Separate proteins

Triton X-114 SDS-PAGE

Separate peptides

Sequence peptides

LC

MS/MS

(b) Glc P P P P

P P

(e)

N-glycosylated membrane proteins

Oxidize N-glycans

Capture N-glycans

Digest proteins

Derivatize/release glycopeptides

Sequence peptides

Periodate

Hydrazide

Trypsin

d0/d4-N-alkylation PNGase F

LC-MS/MS

Glc

GPI GPI

Current Opinion in Chemical Biology

Modification-specific proteomic analysis of membrane proteins: shave and conquer. Membrane proteins are oriented in the plasma membrane, having extracellular domains and intracellular domains. (a,b) The orientation is maintained in membrane vesicles prepared from cellular membrane fractions. (c) Membrane proteins are phosphorylated in the cytosolic domain, which can be exposed by inversion of vesicles by treatment with Brij-58 detergent, allowing proteolytic ‘shaving’ of the phosphorylated domains [16]. Phosphopeptides are subsequently recovered by IMAC and analyzed by LC-MS/MS. (d) GPI-anchored proteins are extracellular proteins that are tethered to the plasma membrane by a GPI-anchor. Cleavage of the GPI-anchor by phosphatidylinositol-phospholipase C (PtdIns-PLC) releases the GPI-proteins, which are then extracted, separated and identified by MS [33,34]. (e) N-glycosylation sites are localized in extracellular domains of membrane proteins (Glc, part b). Being exposed, glycans can be activated by oxidation and thus the vesicles carrying the glycoproteins can be captured by covalent linkage to resins [30]. Glycosylation can be quantified upon proteolysis and derivitization followed by LC-MS/MS analysis of the PNGase F released, isotopically encoded peptides.

a highly active and ‘promiscuous’ protease (proteinase K) to achieve comprehensive degradation of proteins, including cleavage of their hydrophobic domains. Proteinase K generates small, overlapping or staggered peptides that can be separated and sequenced by LC-MS/MS. As a result, each amino acid may be sequenced multiple times as it is part of several different peptides of varying length. This type of ‘shotgun sequencing’ permits reassembly of the amino acid sequence and facilitates determination of post-translationally modified peptides [31,32]. It is however, hampered by the fact that low-abundance posttranslationally modified peptides may be suppressed and escape analysis because of the complexity of the peptide mixture. PTM-specific enrichment methods and MS facilitate a more detailed analysis of modified membrane proteins. This is demonstrated by application of the ‘shave-and-conquer’ strategy (Figure 3), which explores the fact that plasma membrane preparations consisting of right-side-out vesicles expose the extracellular domains www.sciencedirect.com

of membrane proteins. When treated with a protease or a modification-specific enzyme, the released (shaved off) peptides or proteins were amendable to MS. The feasibility of such a targeted modification-specific approach was demonstrated for plant and human glycosylphosphatidylinositol (GPI)-anchored proteins, which were selectively released by phosphatidylinositol-specific phospholipase C and identified by MS [33,34]. In an elegant extension of the shave-and-conquer approach, Nu¨ hse et al. reported the use of the detergent Brij-58 to invert rightside-out membrane vesicles to generate inside-out vesicles [16]. These inverted vesicles expose the cytosolic, phosphorylated domains of membrane proteins. Subsequent ‘shaving’ of the vesicles by trypsin and enrichment by IMAC enabled isolation and sequencing of several hundred phosphopeptides, leading to the identification of many different functional classes of transmembrane proteins in Arabidopsis thaliana and assignment of their phosphorylation sites. Current Opinion in Chemical Biology 2004, 8:33–41

38 Proteomics and genomics

Quantitation of post-translational modifications by mass spectrometry Quantitation of post-translationally modified proteins encompasses not only determination of protein abundance but also relative or absolute determination of site occupancy (i.e. the extent of PTM at each amino acid residue in the protein). Differential stable isotope incorporation by metabolic labeling (in vivo) or by chemical labeling (in vitro) [35,36] enables relative quantitation of PTMs between two or more different states by MS. Metabolic labeling of proteins by growing cell cultures in 15-N containing medium enabled differential (relative) determination of phosphorylation site occupancy in yeast proteins [37]. Addition of stable isotope encoded amino acids such as D3-Leu or 13C6-Arg to the medium [38] and the use of a-pY antibody for phosphoprotein enrichment recently resulted in the determination of modificationdependent protein–protein interactions in the EGF receptor signaling pathway [10], providing new insights into the molecular details of dynamic cell signaling processes (see also Update). Similarly, stable isotope labeling has enabled quantitation of protein degradation processes and the determination of the rate of protein turnover in yeast [39]. Chemical isotope labeling of peptides via belimination/Michael addition has been applied for differential determination of Ser/Thr phosphorylation [21,40]. As mentioned in a previous section, a chemical approach for the capture, identification and relative quantitation of N-glycosylation proved useful for characterization of glycoproteins in serum and in plasma membranes [30]. The addition of stable isotope labeled internal standards facilitates absolute quantitation of modified peptides by selected reaction monitoring MS/MS for targeted analysis of low-abundance, modified proteins and peptides [41]. A general and potentially scalable chemical/biochemical method used N-terminal derivatization and phosphatase treatment for absolute quantitation of phosphorylation [42]. These rapidly evolving quantitative techniques for MS-based determination of PTMs should prove highly useful for studying the dynamics of protein-mediated cellular processes.

Advances in mass spectrometry Mass spectrometers continue to evolve, propelled by demands from the proteomics community for increased sensitivity, higher mass accuracy and resolving power, improved duty cycle and more efficient fragmentation of peptides in MS/MS. Electrospray ionisation MS/MS has been used for several years for peptide sequencing. Generation of diagnostic, PTM-specific reporter ions or fragments by MS/MS using precursor ion scans [24,43,44] or neutral loss scans [45] is an efficient and highly sensitive technique for selective and specific detection of modified peptides, including phosphopeptides and glycopeptides. However, further developments and refinements of sample preparation Current Opinion in Chemical Biology 2004, 8:33–41

techniques, mass spectrometer hardware and software is required to make these scanning methods applicable to the analysis of the highly complex peptide mixtures generated in many proteomics experiments. MALDI MS/MS instruments have now emerged and proven efficient for sequencing of post-translationally modified peptides (e.g. phosphopeptides) [46–48]. The main attributes of MALDI are robustness and high sensitivity and there is an increasing interest in combining MALDI MS/MS methods with miniaturized peptide separation techniques. In situ liquid-liquid extraction is a simple and rapid method for the separation of hydrophobic and hydrophilic peptides before MALDI MS and MS/MS [49]. This method may enable more efficient detection and sequencing of modified, hydrophobic peptides, particularly for acylated peptides. Graphite columns permit capture of otherwise elusive hydrophilic peptides, such as phosphopeptides and glycopeptides, for their analysis by MS [50]. In addition, capillary chromatography and electrophoresis have been interfaced to MALDI MS/ MS in various ways [51]. Because separations, MALDI MS and MS/MS are in effect decoupled in time, this approach allows revisiting previously analyzed fractions, facilitating in-depth analysis of crude peptide mixtures. Fourier transform ion cyclotron resonance (FT-ICR) MS inherently provides very high mass accuracy and resolving power and is bound to make an impact in proteomics. Linear ion traps coupled to FT-ICR improve the duty cycle and data quality attainable in LC-MS/MS by combining efficient ion accumulation and fragmentation with accurate mass determination for improved LC-MS/MS performance [52]. Electron capture dissociation (ECD) FT-ICR MS is a novel technique suited for sequencing of post-translationally modified peptides as it cleaves only the peptide backbone while leaving the modified amino acid side-chains intact [53]. Thus, ECD enables efficient sequencing of phosphopeptides, glycopeptides and other types of modified peptides as well as of intact, modified proteins up to 45 kDa [54,55]. Advances in intact protein separation techniques for MS [56,57] point towards future ‘top-down’ proteomic strategies for determination of PTMs.

Conclusions and outlook PTMs are ubiquitous and dynamic. Their presence cannot be reliably predicted by computational sequence analysis and it is therefore necessary to develop efficient and sensitive experimental proteomic techniques for their determination. Combinations of genetic, biochemical and analytical techniques have already provided a glimpse of the complexity and dynamics of PTMs in ‘the proteome’. Establishing proteomics data standards [58] and distributing information on PTMs in public databases, such as the human protein reference database [59] and SwissProt [60], is highly useful to researchers. No www.sciencedirect.com

Modification-specific proteomics Jensen 39

doubt, the interplay between computational and experimental techniques in proteomics will lead to improved methods for the assignment of modified proteins, peptides and amino acid residues in the future. Integrated computational and MS-driven methods are now used to characterize GPI-anchored proteome in plants [33,34], protein kinase A substrates (Hjerrild M et al., unpublished data) and phosphotyrosine assignments in proteins [61]. Thus, hypothesis-driven experimental approaches for the determination of PTMs are well underway. Nevertheless, qualitative and quantitative determination of PTMs and elucidation of their biological functions will remain a tremendous and exciting challenge in proteomics research for years to come.

Update The feasibility of using a chemoenzymatic approach for mapping of protein phosphorylation sites [25] was previously reported by Hathaway and co-workers [62]. Stable isotope labeled amino acid incorporation in mammalian cell culture has been used as a tool for relative quantitation of phosphopeptides by mass spectrometry [63].

Acknowledgements I thank past and present members of the Protein research group for discussions and for their contributions to protein MS and modificationspecific proteomics. The Danish Natural Sciences Research Council and the Danish Biotechnology Instrument Centre provided research funding. I apologize to those colleagues whose work is not cited due to space constraints.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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Rabilloud T: Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2002, 2:3-10.

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Bykova NV, Stensballe A, Egsgaard H, Jensen ON, Moller IM: Phosphorylation of formate dehydrogenase in potato tuber mitochondria. J Biol Chem 2003, 278:26021-26030.

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Yamagata A, Kristensen DB, Takeda Y, Miyamoto Y, Okada K, Inamatsu M, Yoshizato K: Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase. Proteomics 2002, 2:1267-1276.

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Campbell DG, Morrice NA: Identification of protein phosphorylation sites by a combination of mass spectrometry and solid phase Edman sequencing. J Biomol Tech 2002, 13:119-130.

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Patton WF: Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci 2002, 771:3-31.

10. Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M:  A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 2003, 21:315-318. Stable isotope labeling of cell culture, affinity purification and LC-MS/MS were used to determine phosphotyrosine-dependent interactions in the EGF receptor signaling pathway. 11. Salomon AR, Ficarro SB, Brill LM, Brinker A, Phung QT, Ericson C,  Sauer K, Brock A, Horn DM, Schultz PG et al.: Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc Natl Acad Sci U S A 2003, 100:443-448. Affinity purification of phosphoproteins by immunoprecipitation and of phosphopeptides by IMAC enabled determination of signaling molecules and determination of phosphorylation sites. 12. Gronborg M, Kristiansen TZ, Stensballe A, Andersen JS, Ohara O,  Mann M, Jensen ON, Pandey A: A mass spectrometry-based proteomic approach for identification of serine/threoninephosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate. Mol Cell Proteomics 2002, 1:517-527. Screening and application of anti-phosphoserine/phosphothreonine antibodies for their use in phosphoproteomics. 13. Kane S, Sano H, Liu SC, Asara JM, Lane WS, Garner CC, Lienhard GE: A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J Biol Chem 2002, 277:22115-22118. 14. Stensballe A, Andersen S, Jensen ON: Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. Proteomics 2001, 1:207-222. 15. Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM,  Shabanowitz J, Hunt DF, White FM: Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 2002, 20:301-305. The first large-scale analysis of the yeast phosphoproteome by IMAC and LC-MS/MS techniques. 16. Nu¨ hse TS, Stensballe A, Jensen ON, Peck SC: Large-scale  analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol Cell Proteomics 2003, 2:1234-1243. ‘Shave and conquer’ proteomic strategy targeted towards phosphorylated membrane proteins. Cytoplasmic domains of A. thaliana membrane proteins were exposed via membrane vesicle inversion by Brij-58 detergent followed by cleavage by trypsin. Several hundred phosphopeptides were selectively recovered by SAX and IMAC and then sequenced by LC-MS/MS. 17. Geng M, Zhang X, Bina M, Regnier F: Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests. J Chromatogr B Biomed Sci Appl 2001, 752:293-306. 18. Bunkenborg J, Pilch BJ, Podtelejnikov AV, Wiesniewski JR: Screening for N-glycosylated proteins by liquid chromatography mass spectrometry. Proteomics 2003, in press. 19. Kaji H, Saito H, Yamauchi Y, Shinkawa T, Taoka M, Hirabayashi J,  Kasai K, Takahashi N, Isobe T: Lectin affinity capture, isotopecoded tagging and mass spectrometry to identify N-linked glycoproteins. Nat Biotechnol 2003, 21:667-672. A general glycoproteomics technique based on lectin-affinity enrichment combined with enzymatic 18-O incorporation and LC-MS/MS. Starting from a C. elegans protein extract, hundreds of glycoproteins and their glycosylation sites were determined. 20. Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D, Marsischky G,  Roelofs J, Finley D, Gygi SP: A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 2003, 21:921-926. Using affinity-enrichment of His-tagged ubiquitin as a method to capture conjugated proteins prior to their analysis by SDS-PAGE and LC-MS/MS, this study identified a large set of ubiquitinated proteins in yeast. 21. Goshe MB, Veenstra TD, Panisko EA, Conrads TP, Angell NH, Smith RD: Phosphoprotein isotope-coded affinity tags: Current Opinion in Chemical Biology 2004, 8:33–41

40 Proteomics and genomics

application to the enrichment and identification of lowabundance phosphoproteins. Anal Chem 2002, 74:607-616.

culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 2002, 1:376-386.

22. Oda Y, Nagasu T, Chait BT: Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotechnol 2001, 19:379-382.

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