Modificomics: Posttranslational modifications beyond protein phosphorylation and glycosylation

Biomolecular Engineering 24 (2007) 169–177 www.elsevier.com/locate/geneanabioeng

Review

Modificomics: Posttranslational modifications beyond protein phosphorylation and glycosylation Joerg Reinders a, Albert Sickmann b,* a

University of Wuerzburg, Proteomics Group, Pharmaceutical Biology, Julius-von-Sachs-Institute for Biosciences, Julius-von-Sachs-Platz 2, 97082 Wuerzburg, Germany b University of Wuerzburg, Protein Mass Spectrometry and Functional Proteomics Group, Rudolf-Virchow-Center for Experimental Biomedicine, Versbacher Strasse 9, 97078 Wuerzburg, Germany Received 30 November 2006; received in revised form 6 March 2007; accepted 6 March 2007

Abstract Posttranslational modifications of proteins possess key functions in the regulation of various cellular processes. While they facilitate fast, location-specific and transient reactions to changing conditions in the first place they enhance the already high complexity of a cellular proteome by orders of magnitude. Furthermore, they can utterly alter the properties of the modified protein, thus making a timely analysis even more difficult. While several standardized methods for the analysis of protein phosphorylation and glycosylation have been established most other modifications require tailor-made solutions for a comprehensive analysis. Therefore, we will provide guidelines for the analysis of some important posttranslational modifications that are underrepresented in contemporary literature. # 2007 Published by Elsevier B.V. Keywords: Posttranslational modification; Sulfation; Nitrosylation; Lipidation; Protein oxidation

Contents 1. 2. 3. 4. 5. 6.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of protein sulfation . . . . . . . . . . . . . . . . . . . . . . . Analysis of deamidation . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of protein nitrosylation . . . . . . . . . . . . . . . . . . . . Analysis of protein lipidation . . . . . . . . . . . . . . . . . . . . . . 6.1. Protein prenylation . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Protein acylation . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Glycosyl-phosphatidyl-inositol-anchors (GPI-anchors). Analysis of protein oxidations . . . . . . . . . . . . . . . . . . . . . . 7.1. Oxidation of cysteine residues . . . . . . . . . . . . . . . . . 7.2. Oxidation of methionine residues . . . . . . . . . . . . . . . 7.3. Oxidation of tryptophan residues . . . . . . . . . . . . . . . 7.4. Protein carbonylation . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction * Corresponding author. Tel.: +49 931 201 48730; fax: +49 931 201 48123. E-mail address: [email protected] (A. Sickmann). 1389-0344/$ – see front matter # 2007 Published by Elsevier B.V. doi:10.1016/j.bioeng.2007.03.002

Posttranslational modifications serve many different purposes in various cellular processes such as enzyme regulation, signal transduction, mediation of protein localization, interactions and

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stability. Genomic data can only partly be used for prediction of PTMs although specific software and databases are rapidly evolving (Blom et al., 2004; Chen et al., 2006; Lee et al., 2006; Xue et al., 2006). Therefore, proteomics is the method-of-choice for the analysis of modified proteins and peptides. The enormous versatility of the modifications that frequently alter the physicochemical properties of the respective proteins significantly is only one of the challenges of modification-oriented proteomics. Protein modifications are often transient, substoichiometric, time- and location-specific, site-specific and polymorphic (Sickmann et al., 2002). Thus, the analysis of posttranslational modifications is probably the most versatile and difficult, but also most frequently studied area of interest in proteomics research. This growing field of ‘‘modificomics’’ will yield many important insights into cellular networks but still face further challenges along with analytical and technical progress. Although several hundreds of different modifications are known (Agris, 2004) protein phosphorylations and glycosylations and the respective analysis techniques are more often addressed by contemporary reviews (Harvey, 2005; Morandell et al., 2006; Morelle et al., 2006; Morelle and Michalski, 2005; Mukherji, 2005; Mumby and Brekken, 2005) than other modifications. Nevertheless, essential cellular functions are based on further PTMs such as protein lipidations, nitrosylations, sulfations or oxidative modifications. Thus, we will rather discuss techniques for the analysis of some of these posttranslational modifications that are equally important but have gained less attention during the last years. A comprehensive analysis of different posttranslational modifications in parallel is usually not possible on a global scale. Therefore, the analysis should either be focussed on a single or very few distinct proteins or be directed towards a certain type of modification. Particularly the versatility, stoichiometry and dynamics of protein modifications raise the need for custom-made solutions for each issue to be addressed (Reinders et al., 2004). Therefore, all described methods represent rather general strategies that should be fitted to the respective matter than receipts to be followed step-bystep.

2.

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Modification studies usually require more material than needed for mere identification of a protein often due to low stoichiometry and stability of the modification. But even more urgent than the question of sensitivity are the selectivity of the applied methods, e.g. false-positive identifications, and representativity of the sample, e.g. prevention of artefacts. Will my study be directed at a certain peptide, protein or protein complex or am I trying to accomplish a global overview? Directed approaches deal with samples of reduced complexity and therefore often require affinity purification of the respective subject matter. However, an almost complete view of the modification state may be achieved by in-depth analysis. Many more modifications may be detected by global methods but complexity of the sample will not allow for a complete overview. Can the modified species be separated from the unmodified one by certain techniques? Analysis of modified protein or peptides is usually much easier in the absence of the non-modified species. Thus, such specific enrichment, respectively depletion methods should preferably be used if applicable. Which analysis-depth do I want to achieve? In general, the more detailed the results should be the more work has usually to be invested. Thus, do I want to identify the modified protein, the modified region within the protein, e.g. domain, or the modified amino acid residue? Moreover, is my modification polymorphic and do I want to address the specific type of modification, for instance different types of lipidation. Investigation in the dynamics of the modification usually requires quantitative analysis methods. Do I need qualitative or quantitative data? Obtaining quantitative data is usually more difficult and labour-intensive than providing qualitative information for a specific sample. However, even the time courses of protein modifications may be targeted by quantitative approaches. So, the amount of work that I will need or want to spend should be considered in advance.

2. General considerations Analysis strategies for posttranslational modifications solely depend on the intended purpose of the respective study. So the more you know about your sample and the clearer you can define your aim the bigger are your chances for a successful analysis. The type of posttranslational modification to be analyzed will predefine most of the applicable sample preparation techniques, e.g. by its pH- and solvent-stability, influence on protein solubility or possible occurrence of artefacts. Furthermore, separation and detection methods also depend upon the type of modification. Thus, questions like the following should be clarified in advance: 1. Can I apply appropriate techniques to obtain a representative sample in sufficient amounts?

If these questions cannot be answered unambiguously preexperiments should be carried out to clarify the preconditions and needs preventing experimental mistakes. 3. Analysis of protein sulfation Different types of protein sulfation (O-, S- and N-sulfation) are known (Huxtable, 1986) but sulfation of tyrosine residues occurring almost exclusively on secreted and membranespanning proteins is probably the best studied one (Hille et al., 1984, 1990; Hille and Huttner, 1990; Nemeth-Cawley et al., 2001). Furthermore, tyrosine sulfation is a more frequent modification than the much more thoroughly studied tyrosine phosphorylation (Monigatti et al., 2006) and is similarly thought to facilitate protein–protein interactions. The sulfate moiety is transferred to the hydroxyl group of tyrosine

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sidechains from 30 -phosphoadenosine-50 -phosphosulfate (PAPS) in the trans-Golgi network (Danielsen, 1987). Radioactive labelling using 35S-sulfate is possible both in vitro and in vivo but in vivo-experiments are hampered by endogenous sulfate levels lowering the incorporation rate of radioactive sulfate into PAPS (Monigatti et al., 2006). Thus, relatively high amounts of radioactivity are needed for in vivo studies. Further problems arise from the lack of specific enrichment methods or antibodies and the acid lability of this modification impeding separation and detection techniques involving the use of strong acids such as Edman-degradation. Mass spectrometric means may be used for the analysis of sulfotyrosine but the modification is even more labile than Ser/ Thr-phosphorylation and is often lost using soft ionization techniques such as matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) (Wolfender et al., 1999). However, negative ion mode can enhance sulfopeptidestability as does multiple sulfation of a peptide (Onnerfjord et al., 2004). Consequently, signals originating from the loss of the sulfate-moiety are dominating collisionally induced dissociation-spectra (CID-spectra) of sulfated peptides. Thus, neutral loss scanning (NLS) can be used for identification of sulfopeptides (loss of SO3, Dm = 80 Da) (Salek et al., 2004), but interference from phosphorylated peptides which can yield the same neutral loss (loss of HPO3, Dm = 80 Da), but usually showing a more intensive loss of H3PO4 (Dm = 98 Da), has to be anticipated. The only possibility to distinguish the masses of HPO3 (Dm = 79.98 Da) and SO3 (Dm = 80.06 Da) by mass spectrometry is high resolution Fourier-transform-ion cyclotron resonance- (FT-ICR-) or OrbitrapTM-MS but due to the high cost these instruments are seldom used. Furthermore, localization of the modification to a distinct tyrosine residue within the peptide sequence is usually not possible since the loss of SO3 leaves an unmodified tyrosine. Precursor ion scanning (PIS) of sulfopeptides in negative mode (reporter ion SO3, m/z = 80 Da) is more specific, but unfortunately the reported sensitivity of this method is rather low (Jedrzejewski and Lehmann, 1997). Furthermore, MS/MS-spectra of peptides in negative mode are usually relatively poor compared to positive mode (Sickmann et al., 2003). Electron-capture-dissociation (ECD) can be used for the analysis of sulfated peptides minimizing loss of the sulfatemoiety from the peptide (Haselmann et al., 2001). However, since highly expensive FT-ICR-mass spectrometers are needed this technique is rather seldom applied. In general, the same approach may be accomplished using the recently introduced electron-transfer dissociation (ETD) but up to now no such study has been reported in the literature. Since the usually acidic sulfation motifs in bioactive, mostly neuro-peptides are quite conserved several tools for prediction of sulfation sites are available. For a good overview see Monigatti et al. (2006).

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cause of artificial spots in 2D-electrophoresis. Particularly asparagines that are followed by glycine residues are susceptible to deamidation and are thought to serve regulatory purposes in the cell (Weintraub and Manson, 2004). Deamidation of asparagine and to a lesser extent glutamine side chains can either occur by direct hydrolysis of the amide group or by cyclic imide formation involving an intermediate five- or six-membered ring which can also result in racemization of the amino acid and formation of iso-aspartate (Reubsaet et al., 1998). Conversion of an amido- to a carboxyl-group is accompanied by a +1-Da shift. Therefore, mass-spectrometrical analysis of partial deamidation of a peptide is hampered by the overlying isotope pattern due to 13C-incorporation and only FT-ICR-mass spectrometers provide sufficient resolution to distinguish the – NH2/–OH-exchange from the natural 12C/13C-difference (Dm (– NH2/–OH:12C/13C) = 0.0193 Da) (Schmid et al., 2001). However, deamidated and non-deamidated peptides may be separated and quantified by reversed-phase-chromatography, capillary electrophoresis or isoelectric focussing (Reubsaet et al., 1998). 5. Analysis of protein nitrosylation Nitrosylation of proteins occurs upon modification with reactive nitrogen species such as peroxynitrite (ONOO) or NOx and has been proposed as a marker for oxidative stress in both animal and plant biology (Kim et al., 2002; Schmidt and Walter, 1994; Shapiro, 2005). Proteins may be nitrosylated on cysteine residues leading to the formation of nitrosothiols (–SNO), on tyrosine residues generating 3-nitrotyrosine (–C6H4NO2OH) or on tryptophanes leading to different regioisomers of nitrotryptophan. Viable anti-nitrotyrosine-antibodies are available for both affinity purification and immunoblotting thereby facilitating the detection and quantification of tyrosine modification (Aulak et al., 2004). Furthermore, also quantitative LC–MS-based methods have been established for 3-nitrotyrosine involving complete protein hydrolysis and subsequent LC–MS-measurements in the highly specific multiple-reaction-monitoring (MRM)-mode (Althaus et al., 2000) using m/z = 227.1 Da as parent ion and m/z = 133.1 Da as daughter ion (see Fig. 1). Since reports of antibodies for nitrosocysteine are scarce (Gow et al., 2002) and no LC–MS-methods are known the method-of-choice for the analysis of nitrosothiols is the biotinswitch-method introduced by Jaffrey and Snyder (2001). In this indirect method all free cysteines are blocked by Nethylmaleinimide (see Fig. 2). Afterwards the nitrosothiols

4. Analysis of deamidation Conversion of Asn/Gln to Asp/Glu by deamidation is mostly not occurring during sample preparation and therefore not

Fig. 1. Parent and daughter ion structures as proposed by Althaus et al. (2000). This transition may be used for nitrotyrosine-specific MRM-scanning.

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+46 Da mass-shift (Yamakura and Ikeda, 2006) neutral loss or precursor ion scanning methods have not been reported in the literature so far. 6. Analysis of protein lipidation 6.1. Protein prenylation

Fig. 2. Scheme of the biotin-switch-method (Jaffrey and Snyder, 2001). In the first step all free thiols are blocked, e.g. by N-ethylmaleinimide or methane methylthiosulfonate. Second, sulfenic acid moieties are reduced, nitrosothiols are freed by ascorbate, respectively. In a last step the free thiol-groups are reacted with biotin-HPDP facilitating subsequent affinity purification of the biotinylated proteins and peptides.

are converted into free cysteines by treatment with ascorbate and subsequently reacted with N-[6-(biotinamido)hexyl]-30 -(20 pyridyldithio)propionamide (Biotin-HPDP). The biotin-moiety can then be used for affinity purification of respective proteins and peptides or for immunoblotting. However, unspecific binding to the linker region as well as endogenous biotinylated proteins/peptides may lead to false positive identifications. While generation of antibodies against 6-nitrotryptophan suitable for Western blotting has recently been reported (Ikeda et al., 2007; Yamakura and Ikeda, 2006; Yamakura et al., 1974, 2005) and nitrotryptophan-moieties (see Fig. 3) have been analyzed successfully via LC–MS/MS by observation of a

Protein (iso-)prenylation is a lipid modification attaching farnesyl, dolichol or geranylgeranyl-moieties to cysteine residues close to the C-termini of proteins and often within a conserved motif, the so-called CAAX-box (Roskoski, 2003). These modifications are involved in recruitment of the modified proteins to membranes as well as facilitating protein interactions via prenyl-specific binding domains. For a long time the only method for detection of prenylated peptides was the introduction of radioactive isotopes into mevalonic acid, the biological precursor of isoprenoids (Lai et al., 1990). Thereby, the modified proteins/peptides can be traced throughout the purification and separation process by detection of the radioactivity. However, endogenous levels of mevalonic acid should be lowered – if possible – in order to enhance the incorporation of radioactivity into isoprenoids. In case of cell cultures preincubation of the cells with dialyzed serum and hydroxymethyl-glutaryl-CoA-reductase inhibitors is recommended for at least 1 h. Another approach for the analysis of protein farnesylation by tagging-via-substrate (see Fig. 4) – which is in principle transferable to other prenylations such as geranylgeranylation or also further lipid modifications such as myristoylation (see ‘‘Protein acylation’’) – was introduced by Kho et al. (2004). Thereby, cells are fed with either azido-farnesyl-pyrophosphate or azido-farnesyl-alcohol which does not occur naturally but is readily incorporated into proteins via the protein farnesyl transferase anyway. Subsequently, the azido-moiety is used for conjugation with a biotinylated phosphine capture reagent. By this highly specific reaction a biotin is attached to the

Fig. 3. Different tryptophan modification products induced by nitrating agents and/or ROS. 1: nitrotryptophan, different positions of the nitration are possible including the nitrosylation of the side chain imino group (not shown). 2: hydroxytryptophan, one or two hydroxyl-groups can be attached to different sites within the tryptophan side chain. 3: 3a-hydroxypyrolloindole. 4: N-formylkynurenine. 5: kynurenine. 6: 2-oxindolyalanine.

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label which can subsequently be used for immuno-blotting (Drisdel and Green, 2004) or affinity-purification (Roth et al., 2006a,b). Furthermore, a tagging-via-substrate approach reported for the analysis of farnesylated proteins (Kho et al., 2004) should principally be suitable for acylated proteins as well. In a recent report (Hoffman and Kast, 2006) also ESI-NLS has been used for palmitoylation of cysteines using the loss of C16H30OS (Dm = 272 Da). However, no marker ions were obtained which could have been used for PIS. The neutral loss for N-terminally myristoylated glycine (C14H26O, Dm = 210 Da) was of low-intensity; nevertheless prominent a1- and b1-ions (m = 240 Da/268 Da) were obtained suitable for PIS. 6.3. Glycosyl-phosphatidyl-inositol-anchors (GPIanchors) Fig. 4. Scheme of the tagging-via-substrate-approach. 1: azido-farnesyl-pyrophosphate is fed to the cells and readily incorporated into proteins. 2: the azido-group is specifically modified via the Staudinger reaction using a biotinylated phosphine capture reagent (Biotin-PCR) facilitating subsequent affinity purification.

farnesylated proteins facilitating affinity purification and subsequent identification by mass spectrometry. However, affinity purification using biotin-moieties always bears the risk of false positives (see above). Recently, also direct mass spectrometric techniques have been applied for the analysis of farnesylated peptides using MALDI- and ESI-mass spectrometry (Hoffman and Kast, 2006). While precursor ion scanning (reporter ion C15H25, m = 205 Da) is favourable for ESI-MS, for MALDI-MS neutral loss scanning (loss of C15H24, Dm = 204.3 Da) should be preferred but also peptide fragments can yield these ions, e.g. b2-ion GF (m/z = 204.1 Da), which could lead to false positive results. 6.2. Protein acylation Palmitoylation and myristoylation are the most common types of protein acylation. The fatty acids can be attached to the proteins via either oxyester, thioester or amide linkage often recruiting the respective proteins to membranes (McIlhinney, 1990). Radioactive labelling of fatty acids can be used for the analysis (Navarro-Lerida et al., 2004); preferably positions 9 and 10 should be used for introduction of the label minimizing the chance of b-oxidation and incorporation of the label into other metabolites. Furthermore, preincubation of the cells with sodium pyruvate as an acetyl-CoA-source also diminishes interconversion of fatty acids. The type of fatty acid linkage to the protein can be deduced from its stability in different solvents. 200 mM KOH in methanol will cleave thio- and oxyesters within 1 h while 1 M hydroxylamine–HCl will cleave oxyesters only to a minor extent. Amide linkages are stable under both conditions. Hydroxylamine-cleavage of the thioester-bond has also been used for introduction of a biotin

Glycosyl-phosphatidyl-inositol-(GPI)-anchors are attached to the C-termini of proteins via amide-bonds within the endoplasmic reticulum, thereby anchoring the proteins to the membrane by two fatty acid residues. GPI-anchored proteins are thought to be preferentially located in lipid rafts in both animal and plant cells (Borner et al., 2005; Cordy et al., 2003). Different tools for the prediction of GPI-anchoring of proteins are available based on sequence constraints of the hydrophobic C-terminus of the protein (Antony and Miller, 1994; Eisenhaber et al., 2003). Analysis of GPI-anchored proteins may be facilitated by introduction of radioactivity using tritiated glucosamine allowing subsequent one- or two-dimensional electrophoresis, Western blotting and autoradiography (Gilson et al., 2006) or fluorography (Waterborg and Matthews, 1994). Another method proposed by Elortza et al. (2003, 2006) involves membrane isolation and specific cleavage of the GPIanchor by either phospholipase C or phospholipase D. The released proteins are then separated from the membranes and can be subjected to further analysis. The peptides, e.g. obtained from a proteolytic digest, carrying the remains of the GPIanchor may be furthermore enriched by hydrophilic interaction chromatography (HILIC) and analyzed by MALDI-TOF-(timeof-flight)-MS, for instance (Omaetxebarria et al., 2006). However, the enrichment method is not specific for GPIanchors but highly hydrophilic peptides such as glycosylated species are enriched as well (Hagglund et al., 2004). 7. Analysis of protein oxidations Reactive oxygen species (ROS) are generated upon oxidative stress introducing redox-modifications into proteins which are mostly studied by shifts in 2D-electrophoresis and subsequent mass spectrometry (Sheehan, 2006). Direct oxidation by the most important ROS, the hydroxyl radical OH, results in rather unspecific oxidation of proteins leading to protein inactivation and degradation via the ubiquitin-proteasome pathway (Poppek and Grune, 2006). Other ROS with lower oxidation potential introduce modifications more specifically with important functions in redox signalling (Filomeni and Ciriolo, 2006).

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7.1. Oxidation of cysteine residues

acid is not possible because these modifications are irreversible both in vivo and in vitro.

Cysteine residues may be oxidized both in redox signalling and upon oxidative stress. However, the pKa-values of the sulfhydryl-groups have to be lowered by the surrounding environment to react with oxidants at their biological concentrations (Sheehan, 2006). Thereby, thiols can be oxidized to disulfides, e.g. intramolecular, with other proteins or glutathione (GSH). Diagonal electrophoresis (non-reducing/ reducing) has successfully been applied for the analysis of mixed protein disulfides (Eaton, 2006) if adducts are longer than 10 amino acids. Moreover, methods for the analysis of GSH-adducts involving for instance biotinylated GSH or antiGSH-antibodies have been developed and successfully applied (Dalle-Donne et al., 2003), particularly because GSH is thought to be a direct indicator of the cellular redox status. Furthermore, also direct oxidation of cysteines may occur leading to the formation of sulfenic (–SOH), sulfinic (–SO2H) or cysteic acid (–SO3H), the latter ones being irreversible modifications (Sheehan, 2006). An opportunity for distinction of oxidized and non-oxidized cysteines is given since oxidized thiols do not react with sulfhydryl-specific reagents such as iodoacetamide or maleimide. These can in turn be on their behalf be coupled, e.g. to poly-ethylene-glycol or fluoresceine. Thus, gel shift analysis may indicate cysteine oxidized proteins in comparison to the non-oxidized species. Application of the biotin-switch method (Jaffrey and Snyder, 2001) – similar to the method developed for the analysis of cysteine nitrosylation – has also been reported for the analysis of sulfenic acid modification despite of the high reactivity of sulfenic acid. Application of this method for the analysis of sulfinic or cysteic

7.2. Oxidation of methionine residues Upon oxidative stress methionine residues may be singly or doubly oxidized leading to sulfoxides or sulfones, respectively. Since these modifications are reversible in vivo they play an important role in buffering oxidative stress (Stadtman et al., 2005). However, extreme caution has to be taken (Zhu et al., 2004) because oxidation of methionine is likely to occur during sample preparation or separation by aerial oxygen (Guan et al., 2003) or by metal-catalyzed oxidation on the electrospray tip in ESI-MS (Guzzetta et al., 2002) to an extent of up to 30–50%. On the other hand analysis of peptides containing methionine sulfoxide is facilitated by specific cleavage of oxidized residues upon CID (collision-induced dissociation) generating a neutral loss of methanesulfenic acid (64 Da/CH3SOH). While methionine sulfoxide can thus be distinguished from the isobaric phenylalanine (both residue mass 147 Da) (Sickmann et al., 2001) the low energy fragmentation can hamper peptide identification by hindering further backbone fragmentations. Therefore, a combination of CID for detection of the oxidized methionine and ECD (electron-capture dissociation) for peptide backbone fragmentation has proven useful (Guan et al., 2003). 7.3. Oxidation of tryptophan residues Tryptophan residues are very sensitive to oxidation by ROS and nitrating agents leading to different oxidized and/or nitrated

Table 1 Protein reactive aldehydes generated by glycation and/or lipid peroxidation (Uchida, 2000) Reactive aldehyde

Structure

Derived from

Acrolein

Lipid peroxidation

Malondialdehyde

Lipid peroxidation

4-Hydroxy-2-alkenals (e.g. -hexenal, -nonenal)

Lipid peroxidation

Glyoxal

Lipid peroxidation/glycation

Methylglyoxal

Glycation

Glucosone

Glycation

3-Deoxyglucosone

Glycation

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products (see Fig. 3). Oxidative modifications of tryptophan are especially important because they sensitize the protein for further damage by UV-light (Davies and Truscott, 2001). While viable antibodies are scarce the stability of the oxidized tryptophan-moieties may be analyzed by mass spectrometry, e.g. by LC–MS/MS (Taylor et al., 2003). However, since some of these modifications are isobaric distinction by mass spectrometry is difficult but may be facilitated by application of high-energy-CID. 7.4. Protein carbonylation The introduction of carbonyl groups into proteins can occur directly by either oxidation of amino acid side chains, particularly lysine arginine and histidine, or oxidation of the peptide backbone resulting in backbone cleavage (Ghezzi and Bonetto, 2003). Furthermore, proteins may react with glycation and lipid peroxidation products such as 4-hydroxy-2nonenal or malondialdehyde (Stadtman and Berlett, 1997) (see Table 1). Analysis of carbonylated proteins is mostly accomplished by one- or two-dimensional electrophoresis and subsequent immunoblotting. Thereby, all protein-carbonyls can be targeted by reaction with hydrazides coupled to fluorescent dyes (Ahn et al., 1987), radioactive labels or – which is far more frequent – by reaction with 2,4-dinitrophenylhydrazine (Levine et al., 1990). Dinitrophenyl-(DNP)-adducts can be detected by specific anti-DNP-antibodies that may also be used for immunoprecipitation of carbonylated proteins. Furthermore, a variety of specific antibodies against adducts of lipid peroxidation products are available for immunoblotting (Toyokuni et al., 1995; Yamada et al., 2001, 2004). Unfortunately, these methods can only facilitate identification of the modified protein but no information is obtained regarding the modification site. 8. Concluding remarks Modification-oriented proteomics is one of the fastest growing fields in proteomic research. While various techniques have been established for the analysis of phosphorylation and glycosylation suitable methods for the analysis of other, by no means less important protein modifications have only recently been developed or are still lacking. Thus, such modifications may come into the proteomic research focus even more in the near future bearing valuable information for the understanding of cellular regulation networks. References Agris, P.F., 2004. Decoding the genome: a modified view. Nucleic Acids Res. 32, 223–238. Ahn, B., Rhee, S.G., Stadtman, E.R., 1987. Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized protein on polyacrylamide gels. Anal. Biochem. 161, 245–257. Althaus, J.S., Schmidt, K.R., Fountain, S.T., Tseng, M.T., Carroll, R.T., Galatsis, P., Hall, E.D., 2000. LC–MS/MS detection of peroxynitrite-derived

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