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Review

Phosphoproteomics: Searching for a needle in a haystack

Ales Tichya,⁎, Barbora Salovskaa, Pavel Rehulkab, Jana Klimentovab, Jirina Vavrovaa,Jiri Stulik b, Lenka Hernychovab

a Department of Radiobiology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republicb Institute of Molecular Pathology

,Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic

A R T I C L E I N F O

Abbreviations: ECD, electron capture dissaffinity chromatography; NTA, nitrilotriaceti⁎ Corresponding author at: Department of R

Hradec Králové, Czech Republic. Tel.: +420 9E-mail addresses: tichy@pmfhk.cz, tichya

1874-3919/$ – see front matter © 2011 Elsevidoi:10.1016/j.jprot.2011.07.018

Please cite this article as: Tichy A, et al,j.jprot.2011.07.018

A B S T R A C T

Article history:Received 11 March 2011Accepted 22 July 2011

Most of the cellular processes are regulated by reversible phosphorylation of proteins, whichin turn plays a critical role in the regulation of gene expression, cell division, signaltransduction, metabolism, differentiation, and apoptosis. Mass spectrometry ofphosphopeptides obtained from tryptic protein digests has become a powerful tool forcharacterization of phosphoproteins involved in these processes. However, there is ageneral need to significantly enrich the phosphopeptide content to compensate their lowabundance, insufficient ionization, and suppression effects of non-phosphorylatedpeptides.This paper aims to give a comprehensive overview on the methods involved in recentphosphoproteomics. It presents a description of contemporary enrichment techniques withreferences to particular studies and compares different approaches to characterization ofphosphoproteome by mass spectrometry.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Phosphopeptide enrichmentMS/MSQuantitative phosphoproteomics

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Detection of phosphoproteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Phosphoprotein/phosphopeptide enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1. Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Metal oxide affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.3. Immobilized metal Ion chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.4. On-plate enrichment for MALDI-Ms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.5. Other enrichment methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. MS analysis of phosphopeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.1. The choice of MALDI matrix and ionization techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.2. Direct phosphopeptide sequencing by MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5. Quantitative phosphoproteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

ociation; ETD, electron transfer dissociation; IDA, iminodiacetic acid; MOAC, metal oxidec acid; PSD, post-source decay.adiobiology, Faculty of Military Health Sciences, University of Defence, Třebešská 1575, 500 0173 253 216; fax: +420 973 253 000.@lfhk.cuni.cz (A. Tichy).

er B.V. All rights reserved.

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6. Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 07. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 08. Outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

Genomic era in the end of the last century launched a numberof both prokaryotic and eukaryotic sequencing projects.Obtaining a large number of genomic sequences togetherwith technological advances initiated development of newarea of biological research – proteomics. Its main goal is toelucidate the structure and function of all the products of thegene expression of the given system. It encompasses identi-fication of each of the proteins including their quantification,localization in the cell, determination of protein–proteininteractions, analysis of multi-protein complexes, and char-acterization of post-translational modifications (PTM) such asglycosylations and phosphorylations [1].

Proteinphosphorylation is oneof themost frequent and themost important reversiblemodification innature; it is involvedin many cellular processes such as metabolism, homeostasis,transcriptional and translational regulation, cellular signallingand communication, proliferation, differentiation, apoptosis,and cell survival [2]. Phosphorylation frequently initiates andpropagates signal transduction pathways. It is a transient PTMthat typically leads to changes in conformation, activity, andinteractions of a protein within a very short timeframe [3].

Genomic sequencinghas revealed that 2–3%of all eukaryoticgenes are likely to code for protein kinases, which transferphosphate groups to the substrate, andmore than 1000 humanproteinphosphatases,which catalyze the reverse reaction,havebeen predicted by genome annotation, emphasizing the ubiq-uitous role of protein phosphorylation [4]. Obviously, it isreasonable to invest time and resources in the analysis ofphosphoproteins and thus a new proteomic branch hasemerged – phosphoproteomics. To date, no studies have beenable to clarify how many proteins in eukaryotes are in factregulated by phosphorylation. However, it has been estimatedthatmajority of all proteins arephosphorylatedat somepoint oftheir expression and that more than hundred thousandphosphorylation sites may exist in the human proteome [5]. Itis presumed thatmore than one third of the eukaryotic proteinsisphosphorylated;namelyonserine, threonine, or tyrosinewithapproximate relative ratio 90, 10, and 0.05%, respectively [1].

This paper aims to give a comprehensive overview on themethods involved in the recent phosphoproteomics. It presentsa description of contemporary enrichment techniques withreferences to particular studies and compares different ap-proaches of detection and characterization of phosphopeptides.

2. Detection of phosphoproteins

Regarding the complexity of the goals of phosphoproteomics(i.e. identification of phosphoproteins and phosphopeptides,

Please cite this article as: Tichy A, et al, Phosphoproteomics: Sej.jprot.2011.07.018

determination of the phosphorylation site, and quantificationof phosphorylation) it is not possible to use a single universalmethod, but it is necessary to exploit a wide range ofmethodical approaches. Nowadays there are several strategiesfor characterization of phosphoproteins.

Phosphoproteins can be visualized by Western blottingusing antibodies against general phosphoserine, phospho-threonine, or phosphotyrosine residues [6]. Detection withsuch antibodies provides no information of specific phos-phorylation sites. The protein bands of interest must beexcised from the gels and analyzed bymass spectrometry (MS)for further validation, which can be difficult due to co-migrating proteins. Electrophoretically separated phospho-proteins might be also visualized using a phosphospecificstain such as Pro-Q Diamond [7,8]. This fluorescent stainallows direct, in-gel detection of phosphate groups attached toTyr, Ser, or Thr residues without the need of antibodies. Thestain can be used with standard SDS-polyacrylamide gels anddelivers results in a short time and is fully compatible withdown-stream mass spectrometry. Pro-Q Diamond phospho-protein gel stain is most efficient when used in conjunctionwith SYPRO Ruby total-protein gel stain (both produced byInvitrogen, San Diego, CA, USA) that is quantitative over threeorders of magnitude. Determining the ratio of Pro-Q Diamonddye to SYPRO Ruby dye signal intensities for each band or spotthus provides a measure of the phosphorylation levelnormalized to the total amount of protein. Similar phosphos-pecific staining kit called Phos-tag has been developed, wherea highly selective Zn2+ ion chelator is coupled to a flourophore[9].

One of the most sensitive alternatives available is autora-diography, by which phosphoproteins can be radioactivelylabelled via 32P or 33P and subsequently visualized [10].However, it still suffers some drawbacks as inefficientradioactive labelling due to endogenous ATP pools and cellulartoxicity in vivo, and occurrence of promiscuous phosphoryla-tion events due to the unnaturally high concentration ofkinase compared to the substrate concentration in vitro [5].

MS still remains amethod-of-choice for characterization ofmajority of the phosphorylated proteins. After the proteolyticprocessing, however, it is difficult to analyze phosphopeptidesby MS, especially in the presence of the non-modifiedpeptides. This is mainly due to lower ionization efficiency ofphosphopeptides resulting in their lower signal intensities inthe presence of non-phosphorylated peptide ions. In addition,both the amount of phosphorylated protein as well as thestoichiometry of phosphorylation is often present at lowrelative abundance. Thus, efficient enrichment of the phos-phorylated species (proteins or peptides) prior to MS analysiswill result in increased sensitivity and more efficientcharacterization.

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3. Phosphoprotein/phosphopeptide enrichment

3.1. Immunoprecipitation

The enrichment of molecules with phosphate moiety can beperformed either on level of phosphoproteins or phosphopep-tides. Some groups use immunoprecipitation as a capablemethod for enrichment of tyrosine-phosphorylated proteinsand peptides since it greatly aids in the identification of sites ofphosphorylation. Rush and co-workers [11] used phosphotyr-osine-specific antibodies to enrich tryptic peptides and identi-fied 194 phosphotyrosine sites in hyperphosphorylated Jurkatcells. Steen et al. identified 10 proteins in the epidermal growthfactor receptor signalling pathway using anti-phosphotyrosineantibodies for immunoprecipitation and one-dimensional gelelectrophoresis [12]. More recently, Villén et al. identified 385unique tyrosine phosphorylation sites in mouse liver tissue.Notably, they used as much as 80mg of starting material sincelarge amounts are required for immunoprecipitation [13]. Somestudies employed antibodies to specific motifs in phospho-threonine and phosphoserine peptides [14,15] but many othershave referred thismethod as limited by the antibody specificity[6,16]. Hence, anti-phosphoserine and anti-phosphothreonineantibodies are not yet routinely employed in phosphoproteo-mics due to their low specificity.

3.2. Metal oxide affinity chromatography

In recent years, the development of enrichment techniquessuch as metal oxide affinity chromatography (MOAC) beforeMS analysis has proven beneficial for the isolation ofphosphopeptides from complexmixtures with high specificityand for the determination of sites of phosphorylation [17,18].Titanium dioxide (TiO2) chromatography was introduced inthe last decade, since it was shown to have affinity forphosphate ions from aqueous solutions [19]. Pinkse et al. [20]presented an on-line 2-D/LC strategy for phosphopeptideanalysis using spherical TiO2 particles (Titansphere) in thefirst dimension and reversed-phase (RP) material in thesecond dimension. Under this configuration phosphopeptidesare separated from nonphosphorylated peptides by trappingthem under acidic conditions on a TiO2 column. Trifluoroace-tic acid (TFA) in loading buffer protonates acidic residues andprevents adsorption of nonphosphorylated peptides to TiO2.After washing step, nonphosphorylated peptides flowthrough. Subsequently, phosphopeptides are desorbed (elut-ed) from the TiO2 column under alkaline conditions, separatedon the RP column, and analyzed by nano-flow LC-ESI-MS/MS(liquid chromatography-electrospray ionization-tandemmassspectrometry). NH4OH eluent at pH 10.5 affords a highrecovery of phosphopeptides. The pKa and pKb values forTitania are 4.4 and 7.7, respectively [21], so at high pH, Titaniashould become negatively charged and allow phosphopeptideelution.

The TiO2 enrichment protocol underwent several modifi-cations. For example Larsen et al. [22] changed 0.1 Macetic acidin loading buffer with 0.1% TFA. Recently, a new protocol forphosphopeptide enrichment using TiO2 was introduced. Inthis off-line strategy, peptide loading is performed under

Please cite this article as: Tichy A, et al, Phosphoproteomics: Sej.jprot.2011.07.018

highly acidic conditions using various substituted organicacids including 2,5-dihydroxybenzoic acid (DHB), phthalic acidor glycolic acid to efficiently eliminate binding of nonpho-sphorylated acidic peptides to the TiO2 resin [23,24]. Inaddition, by increasing pH of the elution buffer to 11.3 (usingammonia solution), phosphopeptides are more efficientlyeluted from the TiO2 resin, resulting in increased sensitivity.The same group also provided a protocol to enrich phospho-peptides off-line with Titania-packed pipette tips prior toLC-MS or MALDI-MS analysis [24].

Similarly to TiO2, ZrO2 is positively charged at acidic pH andhas a higher binding affinity towards phosphate than carbox-ylate anions [25]. Moreover, ZrO2 has been used previously as achromatographic resin due to its physical and thermalstability, and thus it is a promising material for phosphopep-tide enrichment. Kweon and Håkansson [26] enriched 100pmol tryptic α-casein and β-casein digests on both TiO2 andZrO2 microtips prior to analysis by negative-ion ESI-FT-ICR(Fourier transform ion cyclotron resonance) MS. They foundmore phosphorylated peptides using ZrO2 and concluded thatTiO2 microtips weremore selective for enrichment of multiplyphosphorylated peptides, whereas the ZrO2 tips enrichedprimarily monophosphorylated peptides probably due toeither the higher acidity of Zirconia or the higher coordinationnumber compared to Titania. Anyway, further investigationfocused on the surface properties of these metal oxides isneeded.Gates et al. [27] recently tested commercially producedpipette tips (Glygen, Columbia, MD, USA) packed with TiO2,ZrO2, and a mixture of both. Their enrichment results indicatethat of the three resins evaluated, the TiO2 one is the mostreproducibly selective for phosphopeptides with the leastnonspecific binding under the buffer conditions used. Inaddition, the data obtained with the Glygen TiO2 tips werebetter in terms of the number of unique sites of phosphory-lation that could be reproducibly enriched in independentexperiments, than those obtained with self-made tips packedwith commercially-available TiO2.

Taken together, TiO2 chromatography provides a highselectivity towards phosphopeptides under the optimizedloading buffer conditions and the off-line setup is simple,fast, and does not require expensive equipment. Furthermore,TiO2 is extremely tolerant towardsmost buffers and salts usedin biochemistry and cell biology laboratories [23]. This hasresulted in a robust method for enrichment of phosphopep-tides, which has already become a highly popular method forlarge-scale phosphoproteomic studies.

3.3. Immobilized metal Ion chromatography

Alternatively, immobilized metal ion affinity chromatography(IMAC) is a widely used affinity based technique for enrich-mentof phosphopeptidesprior toMSanalysis.Metal ions (Fe3+,Al3+, Ga3+, or Co2+) are chelated to nitrilotriacetic (NTA) oriminodiacetic acid (IDA) coated beads, forming a stationaryphase to which negatively charged phosphopeptides in amobile phase can bind [28,29]. Originally, the technique wasused for affinity purification of proteins. It is based on theinteraction of peptides with an N-terminal metal-bindingamino acid such as His, Trp, or Cys, especially when suchresidues are not present in other parts of the molecule [30].

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Andersson and Porath [31] were the first ones, who usedmetal ions for binding of phosphoproteins and phosphoaminoacids.

The major drawback of IMAC is high non-specific bindingof non-phosphorylated peptides with more efficient ioniza-tion resulting in suppression of signal from phosphopeptides.This problem can be partially overcome by O-methyl esteri-fication of the side chain carboxyles [32] resulting in substan-tially improved phosphopeptide enrichment [33], but somehave reported that itmight increase the complexity of analysis[34]. Reduction of the complexity of the peptide samples byisoelectric focusing or ion exchange chromatography [35] canalso help to improve the performance of IMAC as well as pHadjustment [36]. Acidification of the sample prior to IMACprotonates the carboxyl groups on the highly acidic aminoacid residues and reduces nonspecific binding.

An approach employing isotope tagging reagents for relativeand absolute quantification (iTRAQ) was used by Liang et al.[37]to compare commercial and prototypal immobilized metalaffinity chelate and metal oxide resins. They used the Nexustetradentate metal chelator (Valen Biotech Inc., Atlanta, GA,USA) coupled to Dynabeads-MyOne (Invitrogen, Carlsbad, CA,USA) tosylactivated beads (U.S. patent application 20020019496).Their results indicate that prototype Fe3+ chelate resin coupledto magnetic beads is able to outperform either Ga3+-coupledanalogue, Fe3+- or Ga3+-loaded IDA-coated magnetic particles,Ga3+-loaded Captivate beads (Invitrogen), Fe3+-loaded Poros20MC beads (Applied Biosystems, Foster City, CA, USA), or Zr4+-coatedProteoExtract (Calbiochem,SanDiego,CA,USA)magneticbeads. This prototypal magnetic Fe3+ chelate performed com-parably tomagnetic TiO2-coated Dynabeads or commercial TiO2

chromatographic spheres (Glygen), even if the latter were usedwith DHB.

In regards tometal affinity enrichment, a novel Fe3+ chelatematrix based on chelate ligand called PHOS-Select IronAffinityGel (Sigma, St. Louis, MO, USA) should be mentioned, since ithas overcome some of the problems arisingwith IMAC. ClassicIMAC enrichment shows substantial binding of acidic com-pounds thatwasminimized bymethyl esterification accordingto Ficcaro et al. [32], however it leads to difficulties withinterpretation of incomplete esterification and other potentialdegradation processes. PHOS-Select is more specific, thusmethylation is not needed and the manufacturer recom-mended procedure with adjusted pH and optimized time ofincubation resulting in minimal binding of acidic peptides.Moreover, new phosphoprotein enrichment Fe-NTA kit wasintroduced (Thermo Scientific, Pierce, Rockford, IL, USA) andaccording to manufacturer it outperformed PHOS-Select inboth number of total and unique phosphopeptides (862 vs. 430and 178 vs. 90, respectively) [38].

3.4. On-plate enrichment for MALDI-Ms

On-plate enrichment is an advantageous technique thatshould enable minimal sample handling, high-throughput,and lower sample loss than the techniques discussed above.Briefly, it involves simple incubation, rinsing of the sample onthe plate, and addition of matrix [17]. A number of researchgroups have prepared custom MALDI plates employing theprinciples described above or other methods combined with

Please cite this article as: Tichy A, et al, Phosphoproteomics: Sej.jprot.2011.07.018

new technologies. The next section presents recent advancesand a brief overview of some interesting results.

Since MOAC with TiO2 seems to be a very efficientenrichment technique, Lin and co-workers [39] immobilizedTiO2-coated gold nanoparticles to a glass plate and analyzedβ-casein digest by localized surface plasmon resonance. Besidesnonspecific adsorption, they have reported that only a lownumber (18%) of phosphopeptides was detected. Qiao et al.[40]performed an array of sintered TiO2 nanoparticle spotsprepared on a stainless steel plate to provide porous substratewith a very large specific surface and durable functions. Whenβ-casein and protein mixtures were tested, the low detectionlimit, small sample size, and rapid selective entrapmentindicated that this on-plate strategy is quite promising,especially because the β-casein phosphopeptides were stillenriched in the presence of 100-fold excess of bovine serumalbumin (BSA) digest. Zhou et al.[41], who used zirconiumphosphonate monolayers immobilized on porous Siliconobserved excellent selectivity of this approach demonstratedby analyzing phosphopeptides in the digested mixture of β-casein and BSA with molar ratio of 1:100. These are veryimportant aspects of the mentioned studies, since successfulenrichment inpeptidemixtureaddresses the real conditions ofa sample within a complex mixture were nonphosphorylatedpeptides impair ionization.

Recently, Eriksson et al. [42] introduced a novel on-targetphosphopeptide enrichment method where spots consistingof a thin film of anatase (one of the mineral forms of TiO2) aresintered onto a conductive glass surface. The method wastested using β-casein as a model phosphorylated protein aswell as with a custom peptide mixed with its phosphorylatedform. A very low detection limit, a significantly improvedphospho-profiling capability, and a simple experimentalapproach were reported. Wang and Bruening [43] describedan interesting method for modification of Silicon wafers,which serve as MALDI plates with 250 μm-diameter micro-spots of phosphopeptide-binding polymer brushes enclosedby a hydrophobic poly(dimethylsiloxane) layer. Enrichmentresulted in a 5-fold decrease inMALDI-MS detection limits andfemtomole-level sensitivity. They improved enrichment fromsamples that contained a 10-fold molar excess of nonpho-sphorylated peptides with the help of a sonication-assistedrinse and they concluded that this array is attractive foranalyzing femtomole amounts of relatively pure proteins,such as those obtained by immunoprecipitation.

Many other studies prove that on-plate enrichmentstrategy is promising and might provide a powerful tool forthe enrichment, detection, and analysis of phosphopeptides,but we are still waiting for fully functional, efficient, andcommercially available “phospho-chip”, therefore we assumethat “classic” methods as IMAC or MOAC in tips or in-solutionwill be used in the very nearest future.

3.5. Other enrichment methods

Besides the techniques described above, there are variousenrichment approaches like calcium phosphate precipitation,charge-based fractionation (strong anion/cation exchange,hydrophilic interaction chromatography), which are ratherpre-separation techniques used to reduce sample complexity

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prior more specific enrichment methods [29]. Another option ischemical derivatization. Arrigoni et al. showed an approachusing a simple one-step β-elimination/Michael addition reac-tion for the derivatization of phosphoserine and phosphothreo-nine. This modification allows the formation of a uniquefragment ion at m/z 106 under mild collision activationconditions, which can be used for parent (precursor) ionscanning in order to improve both the sensitivity and theselectivity of the analysis [44]. Zhou et al. introduced a multi-step strategy consisting of a sequence of chemical reactionscalled phosphoramidate chemistry (PAC) and usingwhole yeastcell lysate they demonstrated that the method is equallyapplicable to serine-, threonine- and tyrosine-phosphorylatedproteins [45], however the method involves an extensive work-load of many chemical reactions. Detailed description of thesemethods is out of the scope and extent of this review and theyare well covered in recent reviews of Leitner and Lindner [46],Salih [47] or Schmidt et al.[48]. Bodenmiller and co-workersassessed the ability of three common phosphopeptide isolationmethods (PAC, IMAC, and TiO2 affinity chromatography) toreproducibly, specifically and comprehensively isolate phos-phopeptides from a tryptic digest of the cytosolic fraction ofDrosophila melanogaster Kc167 cells [49]. LC-electrospray ioniza-tion MS/MS analysis revealed that each method reproduciblyisolated phosphopeptides. The methods, however, differed intheir specificity of isolation and, notably, in the set ofphosphopeptides isolated, since they detect different, partiallyoverlapping segments of the phosphoproteome. Therefore theauthors concluded that, temporarily, no single method issufficient for a comprehensive phosphoproteome analysis.

4. MS analysis of phosphopeptides

4.1. The choice of MALDI matrix and ionization techniques

After enrichment, phosphopeptides are subsequently (in thecase of MALDI-TOFMS approach) spotted onto a plate. Selectionof matrix crucially affects phosphopeptide signals. Typically,α-cyano-4-hydroxycinnamic acid (CHCA) and DHB are used inphosphoproteomics, but use of other matrices has also beenreported. Surprisingly, 2,4,6-trihydroxyacetophenone (THAP)with diammonium hydrogen citrate (DAHC) was found toovercome suppression of phosphopeptides by the nonpho-sphorylated peptides during positive-ion MALDI-TOF MS analy-sis compared to CHCA [50]. The abundances of phosphopeptidesin tryptic digests of protein kinase C-treated mouse cardiactroponin I were enhanced more than 10-fold and usingTHAP/DHAC led to the identification of a unique phosphoryla-tion site [51]. Kjellström and Jensen [52] have tested severalorganic and inorganic acids as matrix additives to enhancesignal of phosphopeptides in both positive- and negative-ionmodes.After examiningphosphoricacid, formic acid, aceticacid,TFA, and heptafluorobutyric acid they concluded that 1%phosphoric acid added to DHB significantly improved resolutionof MALDI mass spectra of intact proteins. According to Dunn etal.[17] DHB/phosphoric acid typically results in stronger signalthan CHCA. In our hands, DHB/phosphoric acid also yieldsstronger phosphopeptide signals in MS; however, CHCA is moresuitable for MS/MS measurements (unpublished data).

Please cite this article as: Tichy A, et al, Phosphoproteomics: Sej.jprot.2011.07.018

Another soft ionization technique besides MALDI is electro-spray ionization (ESI). Both of these techniques enable thetransfer of intact proteins into the gas phase without fragmen-tation, but that is all these two methods have in common.MALDI produces mostly singly-charged ions and is preferablyused with a high mass range analyzer such as the TOF massanalyzer, while ESI produces multiply charged ions (makinglarger proteins more accessible to analysis than MALDI does)and can be usedwith quadrupoles and ion traps [53]. MALDI is arapid, solid-phase technique that can be utilized for example inhigh throughputmicroarraysor imagingof tissueordetectionofindividual cells or microorganisms. ESI, in contrast, is a liquidtechnique compatible with on-line chromatographic tech-niques and capillary electrophoresis. When coupled with FTmass spectrometers it is more sensitive and reaches highperformance indeed, although the sensitivity of ESI is reducedby the presence of salts, impurities, and organic buffers, whicharemore easily tolerated byMALDI. Interestingly, the ionizationsources of some new FTmass spectrometers (e.g. on the Varianmodel 903, Varian, Palo Alto, CA, USA) are interchangeable, andinclude both ESI and MALDI, because some proteins will onlyionizewith one techniqueor the other (theoverlap is about 30%)and both of them have a place in phosphoproteomics.

4.2. Direct phosphopeptide sequencing by MS/MS

If the site of phosphorylation is to be precisely determined, theexact sequence of phosphopeptide must be known and thespecific modified amino acid must be assigned. Today mostphosphorylation sites are identified by tandem MS, however,sequencing of phosphopeptides by MS/MS is not a trivial taskdue to their low ionization. MALDI-TOF and micro-electro-spray MS instruments have been used for fast identification ofphosphorylated peptides by “signature mass” differencebetween the theoretical and found peptide mass.

To uncover the exact sequence a peptide must be energizedand disrupted into the fragments. If the charge is retained byN-terminal part, the fragment ion is classified according tonomenclature as ion a, b, or c. If it is retained by C-terminalpart, it is classified as ion x, y, or z[54]. Nowadays, frequentlyusedmethod of fragmentation is collision-induced dissociation(CID). In general proteomics it is often performed with triplequadrupole. The first quadrupole selects the ion, which will befragmented (precursor or parent ion). The second one is filledwith an inert gas (usually argon) and the interaction of peptideions andmolecules of collisiongas leads to breakageof apeptidebond.Masses of charged fragments are subsequentlymeasuredby the third quadrupole. TOF–TOFmass spectrometer functionssimilarly; the first TOF analyzer selects precursor ions (whichenter the collision cell located between the two) and thespectrumof fragments ismeasured by the secondTOF analyzer.A combination of a quadrupole and TOF analyzer (so-called Q-TOF) has become very popular; however, in phosphoproteomicsnowadays orbitrap and ion trap instruments play a major roledue to higher sensitivity in full scan MS/MS.

Recently a new hybrid mass spectrometer, the LTQ-Orbi-trap (Thermo, Waltham, MA, USA), was introduced. It consistsof a linear quadrupole ion trap (LTQ) coupled to a novel massanalyzer, the orbitrap, invented by A. Makarov [55]. Theorbitrap mass analyzer employs the trapping of pulsed ion

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beams in an electrostatic quadro-logarithmic field. This field iscreated between an axial central electrode and a coaxial outerelectrode. Stable ion trajectories combine rotation around thecentral electrode with harmonic oscillations along it. Thefrequencies of axial oscillations and hence mass-to-chargeratios of ions are obtained using fast Fourier transform of theimage current detected on the two split halves of the outerelectrode [56]. Importantly the orbitrap is very compact andrequires no magnetic field or special maintenance. LTQ andorbitrap are coupled via the C-trap, an intermediate radiofrequency-only storage device, which can also be used to storebackground ions of known composition. When analyte ionsare added and analyzed together with this “lock mass,” sub-ppm mass accuracy for peptides is achievable [57].

It has been widely demonstrated that during CID in thepositive-ion mode the labile phospho-Ser and phospho-Thrcontainingphosphopeptideswill typically undergoβ-eliminationof phosphoester bond resulting in the loss of phosphoric acid(H3PO4; neutral loss of 98 Da), unlikeTyr residues,which aremorestable and preferentially lose 80 Da (HPO3). On the other hand, inthenegative-ionmodeSer,Thr, andTyr-phosphorylatedpeptidesformphosphopeptide-specificmarker ionsatm/z79 (PO3

−) andm/z63 (PO2

−) [47]. Additionally, scanning for characteristic immoniumion by triple quadrupole instruments was suggested whensearching for Tyr-phosphorylated peptides (m/z 216.043). Steenet al. [58] recommended the use of high resolution MS such asQSTAR Pulsar Q-TOF mass spectrometer (Applied Biosystems,Foster City, CA, USA) to distinguish between a diagnosticimmonium ion and those generated by other a, b, and y ions.

According to the energy used for fragmentation, we candistinguish two types of dissociation; low-energyCID (<100 eV)and high-energy CID (>1000 eV). Low-energy collision activa-tion of phosphorylated peptides, typically leads to the produc-tion of y and b ions and results in the loss of phosphoric acidwith inadequate fragmentation of the peptide backbone andonly limited sequence information is obtained. This can beovercome considerably by using multi stage MS/MS (MS3),where the fragment ion originating from the loss of thephosphate group is selected for a second round of CID [29].Olsen and Mann reported that efficient ion capture in a linearion trap leads to MS3 informative and low-background spectraallowing resolution of ambiguities in identification even atsubfemtomole levels of peptide [59]. A method commonlyknown as Data Dependent Neutral Loss MS3 analysis is a scanmode that improves acquisition of MS3 scans only of thosecompounds that show the desired neutral loss, however, theproduction of neutral loss ions in MS/MS is almost alwaysassociated with partial fragmentation of the precursor ions.These sequence informative fragment ions produced inMS/MSare not included when neutral loss ions are isolated forMS3[60]. A new strategy, termed Multistage Activation (MSA),avoids the loss of sequence-informative ions and providesmore fragments from the ion produced by the neutral loss. Inthis approach, the product ions from both the precursor andthe neutral loss product activation are simultaneously storedand a composite spectrum that contains fragments frommultiple precursors is generated [61]. The utility of dual-stagefragmentation and MSA method for phosphoproteomics wasexamined for instance byNakayasu et al. in analysis of humanpathogen Trypanosoma cruzi[62].

Please cite this article as: Tichy A, et al, Phosphoproteomics: Sej.jprot.2011.07.018

Despite the fact that low-energy fragmentation providesrelatively simple spectra that are easy to interpret, it does notgenerate sufficient collisional velocity to effectively produceimmonium ion fragments, which carry valuable amino acidcomposition information; nor can it cleave the side chainsnecessary to distinguish between the isobaric amino acids[63]. Fragmentation spectra obtained by high-energy CID yieldbesides b and y ions also other types of ions, thus Leu can bedistinguished from Ile (isobaric amino acids) or Gln from Lys(mass difference only 0.036 Da). Notably, more fragment iontypes increase the complexity of the spectrum. Additionally, asimilar term to high-energy CID exists in literature and itcorresponds to “higher-energy CID” also known as “higher-energy C-trap dissociation” or HCD. This term represents aprocess of dissociation, where collision energy is higher thanthat available in ion traps, but it is still lower than that used forhigh-energy collisions in TOF/TOF or magnetic instruments.

Savitski et al. reported that different fragmentation tech-niques differ strongly in their ability to localize phosphorylationsites [64]. At 1% false localization rate, the highest number ofcorrectly assigned phosphopeptides was achieved by higher-energy CID in combination with an Orbitrap mass analyzerfollowed very closely by low resolution ion trap spectra obtainedafter ETD (see below). Gant-Branumand colleagues have shownthat usingmultiple proteases (trypsin, chymotrypsin, andGluC)and replicate experiments of LTQ- and LTQ-Orbitrap-MS,yielded combined sequence coverage of membrane-associatedadaptor protein APPL1 greater than 99% [65].

Another option for detection of phosphopeptides is so-called post-source decay (PSD) which also takes advantage ofthe phosphorylation-specific losses. The molecular ion ofinterest is selected by ion gate and undergoes “post-source”decay in the first field-free region of the instrument. MALDI-PSD can be performed on MALDI-TOF instruments equippedwith reflectron, which detects the correct mass based onkinetic energy. Observation of the loss of 98 or 80 Da from themolecular ion is diagnostic for phosphorylated peptides andcan be used to distinguish between serine/threonine andtyrosine phosphorylation and in many cases can be used todetermine the exact site of phosphorylation [66]. Neverthe-less, this technique is in recession mainly due to generalavailability of TOF/TOF instruments and time-consumingaspects which has definitely relegated PSD.

A complementary MALDI-TOF method convenient forphosphopeptide detection is dephosphorylation by the treat-ment of peptideswith alkaline phosphatase [67]. This typicallyresults in 80 Da shift to lower mass compared with thepreviously phosphorylated peptide. Thus, it is possible toidentify candidate phosphopeptides by comparison of MALDIspectra recorded before and after enzymatic modification ofthe analyte, and subsequent search for peaks that disappearfrom the treated sample, as well as peaks that appear orincrease in intensity.

Alternatively, electron capture/transfer dissociation (ECD/ETD) can be employed since these methods primarily yieldfragment ions originating from the peptide backbone fragmen-tationwithout concomitant loss of the phosphate group [1]. ECDwas developed by Zubarev and colleagues [68] as amethod thatinvolves the direct introduction of low energy electrons totrapped gas phase ions. It has the remarkable and useful ability

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to generate odd-electron, free-radical driven fragmentation ofthe same type as are generated by electron impact MS. Forproteins, ECD cleaves between the backbone amide and thealpha carbon to produce significantly different types of frag-ment ions than CID (primarily c and z ions). However, Liu andHåkansson [69] found out that abundant b ions are also formedin ECD of peptides without basic amino acid residues and theyproposed that such ionsare formedas a consequenceof protonsbeing located at backbone amide nitrogens. Labile post-trans-lational modifications such as phosphorylation (and O/N-glycosylation or others) remain attached to thebackboneduringanalysis allowing identification and determination of the site.The main advantage of ECD in the context of phosphoproteo-mics is the possibility of highly efficient fragmentation ofmultiphosphorylated peptides. The major drawback is thatECD method is used on rarely accessible instruments such asFT-ICR mass spectrometers (due to requirement of a staticelectromagnetic field for the thermal electrons), althoughrecently some investigators have indicated that it has beensuccessfully used in an ion trap mass spectrometer [70].

When comparing merits of CID and ECD for phosphopep-tide identification andphosphorylation site localization, linearion trap CID was shown to be most efficient for phosphopep-tide identification, whereas FT-ICR ECD was superior forlocalization of phosphorylation sites [71]. Anyway, in the lastyears a substantial progress has been made to find ECD-likedissociation method in the lower-cost instruments. Syka andco-workers [72] invented ETD, which induces fragmentation ofpeptides by transferring electrons to them. Unlike ECD, whichuses free electrons, ETD employs for this purpose radicalanions (e.g. anthracene or azobenzene). ETD cleaves randomlyalong the peptide backbone (yielding c and z ions), while sidechains and modifications such as phosphorylation are leftintact. The technique is satisfactory only for higher chargestate ions (z>2), however compared to CID, ETD is optimal forthe fragmentation of longer peptides or even entire proteins.Thismakes the technique important for top-downproteomics.Importantly, an alternative protease such as Lys-C might beused since it hydrolyzes specifically at the carboxyl side of Lys,thus creating longer peptides [29]. Despite of the fact thatsignal-to-noise ratio of spectra obtained in ETD isusually lowerthan those obtained in CID, the number of fragmentationspectra and the coverage of the sequence is greater in ETD.Taken together, since both CID and ETD are complementaryand compatible fragmentation techniques, it is advised tocombine both approaches in order to elucidate phosphopep-tide sequence [73]. Naturally, it is recommended to apply firstof all an appropriate enrichment technique such as IMAC orMOAC [18,74].

5. Quantitative phosphoproteomics

The technical advancements of the last decade now facilitatethe large-scale identification studies and thousands of phos-phorylation sites can be identified within a fewweeks. But notonly identification but also quantification of phosphorylationis one of the pillars of phosphoproteomics. In this section, themethods necessary for comprehensive and quantitativemeasurement of cellular signalling pathways modulated by

Please cite this article as: Tichy A, et al, Phosphoproteomics: Sej.jprot.2011.07.018

phosphorylation are described. Most of these quantificationmethods rely on the incorporation of heavy stable isotopes toproduce identically-behaving peptides withmodest differencein mass. Label-free approaches have also been utilised forphosphopeptide quantification.

Among the many formats for quantitative proteomics,stable-isotope labelling by amino acids in cell culture (SILAC)has been proven as a simple and powerful one [75]. Thisapproach involves metabolic incorporation of labelled aminoacids into all proteins from cells of one population andsubsequent combination of labelled and unlabelled samplesin equal ratios. During MS analysis, the peptides are detectedas pairs that are spaced by the number of heavy isotopes ineach heavy amino acid the peptide carries. These two signalsenable quantification through the integration of extracted ionchromatograms [76], whichwas successfully exploited e.g. forquantitative analysis of the human spindle phosphopro-teome at distinct mitotic stages and provided insight intophosphorylation dynamics during mitosis [77] or to revealquantitative site-specific changes in tyrosine phosphoryla-tion induced by CSF-1R tyrosine kinase activation andidentify many candidate SRC-dependent substrates phos-phorylated downstream of receptor tyrosine kinases inepithelial cells of breast and lung cancer patients [78].Recently, a detailed phosphoproteomic SILAC analysis ofFLT3 signalling (an attractive drug target) has been performedin human acute myeloid leukaemia cells, again by Villén–Gygi's group, which contributed greatly in this area [79].Obviously, SILAC attracted attention of many scientists andapart from other aspects it does not lead to false positives asprotein-interaction studies, discovers large-scale kinetics ofproteomes, and reveals key issues in the regulation of themolecular signalling within a cell [75].

An alternative to metabolic labelling that can be performedwithout growing cells or organisms in special conditionsinvolves isotope tagging reagents for relative and absolutequantification (iTRAQ; Applied Biosystems, Foster City, CA,USA) [80] or tandem mass tags (TMT; Proteome Sciences,Franfukt am Main, GER) [81]. Peptides are labelled withcommercial isobaric chemical tags, which avoid mass hetero-geneity for the parent ions and rather rely on quantification ofintense low-massMS/MS signature ions of the reporter groupsreleased from the tag during CID [82]. The reporter ions thatcan be used to relatively quantify the peptides and the proteinsfrom which they originated using software such as the freelyavailable i-Tracker [83]. Nowadays, up to eight differentproteomic samples can be labelled using eight differentisobaric tags. Any peptide labelled with the tag has the samenominal mass, an important characteristic that provides asensitivity enhancement over mass-difference labelling. Bojaet al. assessed relative changes in protein phosphorylation inthe mitochondria upon physiological perturbation usingiTRAQ labelling and LTQ-Orbitrap with HCD capability [84].Zhang et al. used optimized Orbitrap-HCD approach andreported a dynamic range of two orders of magnitude foriTRAQ quantification of phosphopeptides in FLT-3-kinase-mutated model system of human myeloid leukaemia [85].

The application of isobaric tags circumvents severalproblems encountered when using isotope-coded affinitytags (ICAT), one of the earliest-developed methods, which

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was introduced by Aebersold and colleagues [86]. Proteinsextracted from the two samples are labelledwith either light orheavy ICAT reagents, and react via cysteinyl thiols on theproteins; but the method can be expanded to any residue towhich a tag can be conjugated. The tag contains an iminobiotinmoiety so the peptides are recovered by avidin affinitychromatography and are subsequently analyzed by LC-MS/MS. However, this analysis suffers from some drawbacks.For example, variable retention times for the same peptide canoccur from deuterium incorporation [87], which was circum-vented by the second-generation ICAT reagents with 12C and13C isotopes. The iminobiotin affinity tag can also be problem-atic due to excessive labelling or any endogenous biotin in thesample matrix (e.g. serum) reducing the column affinity andICAT peptides are sometimes isobaric with eluted non-taggedpeptides, leading to false-positive and false-negative detec-tions, and most importantly, potential under-representationof proteins may occur [88].

Another strategy of quantitative phosphoproteomics is theabsolute quantification (AQUA), which was presented byGygi's lab [89]. This method is based on a common principleof internal standards. The peptides are synthesized withincorporated stable isotopes to mimic native peptides formedby proteolysis. These synthetic peptides can also be preparedwith covalent modifications (e.g. phosphorylation, methyla-tion, acetylation, etc.) that are chemically identical to natu-rally occurring post-translational modifications. Such AQUAinternal standard peptides are then used to precisely andquantitatively measure the absolute levels of proteins andpost-translationally modified proteins after proteolysis byusing a selected reaction monitoring analysis (SRM) in atandemmass spectrometer (primarily triple quadrupole). Thisapproach is typically limited to targeted analysis of specificproteins, such as the recent determination of the phosphor-ylation stoichiometries of two tyrosine residues in the proteintyrosine kinase Lyn in transfected human HEK293T cells andtwo cultured human multiple myeloma strains [90] orcharacterization of the site-specific dephosphorylation of thecell polarity protein Par 3 by protein phosphatase 1α [91].

Unlike the methods described above, label-free quantifica-tion does not use a stable isotope containing compound to tagtheprotein. Label-free approach relies on the changes inanalytesignals directly reflecting their concentrations in one samplerelative to another. This technology employs overall spectralintensity normalization by interpreting signals of moleculesthat donot change concentration fromsample to sample.Usageof high-resolution mass spectrometers is inevitable as well asimplication of specific software. Label free approach is wellcovered in the review of Bantscheff et al. [92] and a compre-hensive comparison with similar method (spectral counting)and SILAC was published by Asara and co-workers [93].

6. Bioinformatics

Data analysis and interpretation remain major challengeswhen attempting to identify large numbers of proteinsequences. In regard to the large number of mass spectrom-etry data, search engines should be used to facilitate thisprocess.

Please cite this article as: Tichy A, et al, Phosphoproteomics: Sej.jprot.2011.07.018

Amethod for performing protein identification and peptidesequencing by utilizing mass spectrometry fragmentationpatterns to search protein and nucleotide databases hasbeen developed in the lab of John Yates [94]. Their programSEQUEST converts the character-based representation ofamino acid sequences in a protein database to fragmentationpatterns which are compared against the MS/MS spectrumgenerated on the target peptide. The algorithm initiallyidentifies amino acid sequences in the database that matchthe measured mass of the peptide, compares fragment ionsagainst the MS/MS spectrum, and generates a preliminaryscore for each amino acid sequence. A cross correlationanalysis is then performed on the top 500 preliminary scoringpeptides by correlating theoretical, reconstructed spectraagainst the experimental spectrum. Output results are dis-played accordingly. In other words, SEQUEST performsautomated peptide/protein sequencing via database search-ing of MS/MS spectra without the need for any manualsequence interpretation, though it can make use of inter-preted sequence information if available.

Another powerful search algorithm that is able to identifyproteins from primary sequence databases is Mascot (MatrixScience, Boston, MA, USA). It features: i) Peptide MassFingerprint, where the experimental data are a list of peptidemass values froman enzymatic digest of a protein ii) SequenceQuery, where one or more peptide mass values are associatedwith information such as partial or ambiguous sequencestrings, amino acid composition information,MS/MS fragmention masses, and-so-forth, and iii) MS/MS Ion Search, which isidentification based on raw MS/MS data from one or morepeptides [95].

Sequest, Mascot or other programs often successfullyidentify the proper peptide sequence, but they fail to provideinformation about the presence or absence of site-determiningions. As a result, one must manually inspect each spectrum toconfirm proper site localization. Gygi's lab presented a proba-bility-based score, named the Ascore, which measures theprobability of correct phosphorylation site localization based onthe presence and intensity of site-determining ions in MS/MSspectra [96]. It re-analyzes phosphopeptide search engineresults to assign a confidence value to each phosphorylatedsite. Similarly, PhosphoScore (an open-source assignmentprogram that is compatible with phosphopeptide data frommultiple MS levels) is an algorithm that takes into account boththematchquality andnormalized intensity of observed spectralpeaks compared to a theoretical spectrum. Ruttenberg et al. [97]reported that PhosphoScore produced >95% correct MS/MSassignments from known synthetic data, >98% agreement withanestablishedMS/MSassignment algorithm(Ascore), and>92%agreement with visual inspection of MS3 and MS4 spectra.Additionally, Mascot Delta Score (MD-score) was validated andachieved similar sensitivity and specificity for phosphositelocalization as the Ascore, which is mainly used in conjunctionwith Sequest.

Nowadays, there are several databases gathering informa-tion about phosphorylation sites. One of these is PHOSIDA(http://www.phosida.com), a phosphorylation site database,which in these days comprises more than 80,000 phosphory-lated, N-glycosylated or acetylated sites from nine differentspecies [98]. For each phosphosite, PHOSIDA lists matching

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kinase motifs, predicted secondary structures, conservationpatterns, and its dynamic regulation upon stimulus. Usingsupport vector machines, PHOSIDA also predicts phosphosites.Another one is the Phospho.ELM that currently comprises over40,000 Ser, Thr, andTyrnon-redundantphosphorylation sites. Ithas features such as structural dis/order, accessibility informa-tion, and a conservation score. Special emphasis has been puton linking to external resources such as interaction networksand other databases [99].

7. Conclusions

The need to effectively perform phosphoproteomics is of agreat interest and it is driven by the need to characterize thephosphate “on/off” phenomena since it is essential to aplethora of biological regulatory processes. Gathering infor-mation about phosphorylation helps to understand theintricate signalling pathways and cell networks. Furthermore,phosphorylation affects important factors that influenceprotein function such as conformation, activity, protein–protein interaction, and so forth.

Unlike general proteomics, where protein identification bymass spectrometry combined with bioinformatics is usedroutinely and effectively, phosphoproteomics remains chal-lenging and the identification of phosphorylation sites re-quires specific approaches.

It is likely that due to the complexity of the goals ofphosphoproteomics it is not possible to use a single universalprotocol, but it is necessary to employ a variety of methods.However, each approach described above has advantages anddisadvantages, and it is up to the scientists to decide whichapproach will be used according to the available instrumentalconditions, the aims of the study, and last but not least theskills of the experimenter. Certainly, minimal sample han-dling and lower sample loss is crucial. For instance, with aconventional IMAC technique, 10–15% of phosphopeptideswere lost during the rinsing step, an additional 10–20% wasstill retained on the IMAC column after elution, and another10–20% of the phosphopeptides were lost when samples weredesalted with C18 material [100].

Therefore it is important to keep a phosphoproteomicstrategy simple with relatively few sample preparation stepsin order to maximize the sensitivity. When complex samplehandling is inevitable, high amount of starting material (ifavailable) is recommended. Important aspectof thesuccessfullyperformed enrichment is sample preparation and the use ofappropriate cocktail of phosphatase inhibitors is essential.

8. Outlook

In the future we expect more investigations in the field ofenrichment and especially recovery of isolated phosphopep-tides during washing, elution, desalting etc. MOAC and IMACare recently the methods of choice, since antibody-basedisolation is not yet accepted as a general technique. On theother hand, together with development of new surfaces andmaterials “on-plate” enrichment seems very promising. Weanticipate an innovative platform for automated phosphopro-

Please cite this article as: Tichy A, et al, Phosphoproteomics: Sej.jprot.2011.07.018

tein isolation and hopefully a commercial, fully functioningphospho-enrichment protein chip will be available.

Besides low-cost instrumental equipment, which will beaccessible more routinely, we expect that in the future,improved databases with information on published PTM willgreatly facilitate effective data representation (a crucial part ofany high-throughput analysis). Moreover, the growingamount of data from large-scale phosphorylation studieswill enable improvements in algorithms for computationalprediction of phosphorylation and thus phosphoproteomicswill be no more a search for a needle in a haystack.

Acknowledgements

This work was supported by the Ministry of Defense of CzechRepublic (projects MO0FVZ0000501 and FVZ0000604). Theauthors report no conflicts of interest. The authors alone areresponsible for the content and writing of the paper.

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