Research article

Journal of

MASS SPECTROMETRY

Received: 31 December 2014 Revised: 23 February 2015 Accepted: 24 February 2015 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3586

High-throughput workflow for identification ofphosphorylated peptides by LC-MALDI-TOF/TOF-MS coupled to in situ enrichment onMALDI plates functionalized by ion landingLukáš Krásný,a,b Petr Pompach,a,c Marcela Strnadová,a Radovan Hynek,b

Karel Vališ,a,c Vladimír Havlíček,a,d Petr Nováka,c and Michael Volnýe*

We report anMS-based workflow for identification of phosphorylated peptides from trypsinized protein mixtures and cell lysatesthat is suitable for high-throughput sample analysis. The workflow is based on an in situ enrichment on matrix-assisted laser

desorption/ionization (MALDI) plates that were functionalized by TiO2 using automated ion landing apparatus that can operateunsupervised. The MALDI plate can be functionalized by TiO2 into any array of predefined geometry (here, 96 positions for sam-ples and 24 for mass calibration standards) made compatible with a standard MALDI spotter and coupled with high-performanceliquid chromatography. The in situ MALDI plate enrichment was compared with a standard precolumn-based separation andachieved comparable or better results than the standard method. The performance of this new workflow was demonstrated ona model mixture of proteins as well as on Jurkat cells lysates. The method showed improved signal-to-noise ratio in a single MSspectrum, which resulted in better identification byMS/MS and a subsequent database search. Using the workflow, we also foundspecific phosphorylations in Jurkat cells that were nonspecifically activated by phorbol 12-myristate 13-acetate. These phosphor-ylations concerned the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and its targetsand were in agreement with the current knowledge of this signaling cascade. Control sample of non-activated cells was devoidof these phosphorylations. Overall, the presented analytical workflow is able to detect dynamic phosphorylation events inminimally processed mammalian cells while using only a short high-performance liquid chromatography gradient. Copyright ©2015 John Wiley & Sons, Ltd.

Additional supporting information may be found in the online version of this article at the publisher’s web site.

Keywords: phosphopeptides; MALDI; enrichment; cell lysates; automation

* Correspondence to: Michael Volný, Applied Physics Laboratory, University ofWashington, Seattle, WA 98105, USA. E-mail: volny@uw.edu

a Institute of Microbiology ASCR, v.v.i., Vídeňská 1083, Prague 142 20, Czech Republic

b Institute of Chemical Technology, Technická 5, Prague 16628, Czech Republic

c Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8,Prague 128 40, Czech Republic

d Regional Centre of Advanced Technologies and Materials, Faculty of Science,Palacky University, 17.listopadu 12, Olomouc 771 46, Czech Republic

e Applied Physics Laboratory, University of Washington, 1013 NE 40th St, Seattle,WA, 98105, USA

1

Introduction

Protein phosphorylation, a covalent attachment of a phosphategroup, was described more than 50 years ago,[1] and it is nowconsidered to be one of the most important mechanisms of cellsignalization and regulation of protein functionality.[2] Proteinphosphorylation and dephosphorylation are mediated by proteinkinases and phosphatases and are involved in a large number ofbiological processes such as signal transduction, molecular recogni-tion and other molecular interactions.[3,4] The dysfunction ormisregulation of these processes is often connected with a widerange of diseases,[5,6] and elucidation at the molecular level, includ-ing the characterization of the protein phosphorylation sites, isrequired for a better understanding of the linked disease states.

MS, which entered the field of proteomics soon after softionization techniques were discovered in the late 1980s, is alsosuccessfully used for identification of protein phosphorylation.Unfortunately, detection of phosphorylated peptides by MS ishindered by low occurrence of phosphorylation (only a fraction ofprotein molecules might be phosphorylated in vivo) and by signalsuppression effects[7] during ionization. Harder ionization may leadto the cleavage of the bond between the phosphate group andaminoacyl residue resulting in the loss of information about the

J. Mass Spectrom. (2015)

phosphorylation status. Isolation, preconcentration and differentchromatographic separations after enzymatic digest are oftenutilized to overcome these difficulties in phosphopeptide analysis.Enrichment of phosphopeptides prior to analysis is a frequently usedstep, and various methods compatible with MS have been devel-oped. Most common approaches include immobilized metal affinitychromatography,[8–10] metal oxide affinity chromatography[11,12] ordifferent chemical modification strategies.[13,14] Comprehensive

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reviews of the phosphoenrichment techniques were presented byDunn et al.[15] and by Leitner et al.[16]

Matrix-assisted laser desorption/ionization (MALDI) MS is apopular MS ionization technique, mainly for its speed and for therelative tolerance to complex sample matrices. Methods havealso been introduced, which simplify the sample pretreatment bythe use of special MALDI plates functionalized by differentsorbents. Such plates can be then used for direct in situ enrichment(selective preconcentration) and subsequent MALDI ionizationand detection.[17] This approach, sometimes referred to as the‘lab-on-plate’,[17] enables manipulation of very small samplevolumes as well as reduction of sample treatment to a few simplewashing steps. Another example of the on-plate sample treatmentis the immunoMALDI technique, which is based on immunoaffinityinteractions.[18,19]

Various modification techniques of MALDI target plates havebeen reported for on-the-plate phosphopeptide enrichment.[20–26]

TiO2 nanoparticles were simply sintered on the MALDI plate asproposed by Qiao et al.[25] Nanostructured TiO2 layer was mountedby pulsed laser deposition.[22] The TiO2 gel was also smeared on theconductive glass slide,[21] and ultraviolet light was used tophotocatalytically pattern TiO2.

[24] More recently, Chen et al.[26]

reported coating of the MALDI target by a polydimethylsiloxanenanofilm with immobilized titanium dioxide nanoparticles. A previ-ous report compared selected published approaches based on thenumber of phosphopeptides detected from a standard mixture oftrypsinized casein.[23]

It has been shown previously that MALDI plates can be modifiedby ion reactive landing to obtain functionalization by TiO2 orZrO2.

[20,27] Although reactive landing of ions in vacuum can achievesome unique modifications of metal surfaces,[28] it suffers fromrelatively low efficiency.[29] To overcome this limitation of ionlanding in vacuum, the ion deposition experiment at atmosphericpressure was studied by Badu-Tawiah et al.[30][31] and named ‘ambi-ent soft landing’. We have subsequently developed atmosphericpressure ion landing into a method for modifications of MALDIplates by TiO2 and ZrO2 for in situ phosphopeptide enrichmentprior MALDI ionization[23] and reported that ion landing is a fast,non-expensive and reproducible technique for MALDI targetfunctionalization. TiO2 and ZrO2 spots prepared by ion landing atatmospheric pressure have large affinity toward the analyte, forma very durable bond with the MALDI target and are stable duringthe entire experiment. In addition, using this technique, it ispossible to modify a large variety of MALDI targets including

Figure 1. Scheme of the apparatus for surface modification by ion landingautomated for array deposition (adapted from Krasny et al.[23]). MALDI, matrix-a

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stainless steel, aluminum, gold, indium-tin oxide glass or conduc-tive polymers [polymeric anchor chips (PAC)].[23]

In the current work, we demonstrate that MALDI targets can bemodified by depositing an array of TiO2 spots (of predefined geom-etry). Fast in situ enrichment on the plate and hyphenation withliquid chromatography (LC) systems are easily possible. That allowsmore efficient and automated sample handling and thus muchhigher sample throughput. Coupling with chromatographicseparation also allows introducing more complex protein samples,including trypsinized cell lysates.

Experimental

Apparatus for modification of MALDI plates

The lab-made automated apparatus for ambient ion landing (Fig. 1)was reported previously.[23] Briefly, a modified electrosonic spraysource was used as the electrospray emitter. The sprayer was basedon a standard one sixth stainless steel T element (Swagelok) andwas assembled using standard fittings, with stainless steel, graphiteand polyether ether ketone ferrules. Fused silica was used for tub-ing. A syringe pump (KD Scientific) was used for pumping the solu-tions into the sprayer. Electrospray was established between thesprayer and the grounded heated desolvation tube by applying ahigh voltage (3 kV) on the metallic part of the syringe. Desolvatedions passed through slit mask that defines the spot size and landedon the MALDI plate. The stage with vertically mounted MALDI platewas moved by two stepper electromotors and controlled by a re-mote console. High voltage (3–4 kV) of the opposite polarity withrespect to the spray voltage was connected by a clip to the MALDIplate. An in-house written software, which controlled the appara-tus, allowed us to create user-defined preprogrammed arrays witha defined number of rows, columns, position coordinates, sprayingdistance and spraying time per position. This significantly improvedautomatization of the MALDI plate modification process, and as aresult, the apparatus could be left run unattended.

Preparation of modified PAC MALDI plates

Polymeric anchor chip plates (Bruker Daltonics) were washed withacetonitrile and water to remove prespotted alpha-Cyano-4-hydroxycinnamic acid (CCA) matrix. A total of 96 TiO2 spots(8 rows and 12 columns) of 2-mm diameter was deposited ontoPAC using the previously described apparatus by ion landing.

at atmospheric pressure. The motion of the target is preprogrammed andssisted laser desorption/ionization.

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For each spot, 50-mM solution of titanium(IV) n-propoxide inisopropanol was sprayed for 5min per position at the flow rateof 250μl/hod; the desolvation tube was heated to 130 °C. An-other array of additional 24 spots (four rows and six columns)of 1-mm diameter was deposited among the already depositedspots. This superimposed array was used for calibration stan-dards. The overall plate modification time was 10h. This couldbe considered inefficient, but it needs to be pointed out thatthe apparatus does not need to be supervised and that theoverall time could be easily decreased by multiplexing the wholeprocess (e.g. by using multiple sprayers or parallel depositions).

Chemicals and materials

Water and acetonitrile (ACN) were purchased from Merck(Germany). MALDI matrices and calibration standard were pur-chased from Bruker Daltonik (Germany). T-cell lymphoma Jurkatcell line (clone E6.1) was obtained from ATCC collection (ATCC,USA). RPMI1640 medium with L-glutamine were from Lonza (CzechRepublic). Fetal bovine serum was from Gibco (UK), and penicillinand streptomycin were from GE Healthcare (Germany). All otherchemicals were purchased from Sigma-Aldrich.

Standard proteins

The three standard proteins for model mixture were obtainedfrom Sigma-Aldrich. Bovine serum albumin (BSA) is not phos-phorylated and was used to model the protein matrix. Caseincomposes of three phosphorylated subunits α-S1 casein, α-S2 ca-sein and β-casein. Ovalbumin from the chicken egg white is aphosphorylated glycoprotein.

Sample treatment (standard protein mixture)

Model mixture of BSA, ovalbumin and casein (1 :1 : 1) at the total con-centration of 1mg/mlwas reduced by Tris(2-carboxyethyl)phosphine(final concentration of 10mM in sample), alkylated by iodoacetamide(final concentration of 30mM in sample), and digested overnight bytrypsin (Promega) at 37 °C. Resulting peptides and phosphopeptideswere desalted using MicroTrap columns (Michrom), dried and dis-solved in solvent A [2% acetonitrile (ACN)/0.1% trifluoroacetic acid(TFA)] to final concentration of 0.05mg/ml for high-performanceliquid chromatography (HPLC).

3

HPLC (standard protein mixture)

Five microliter of the sample (corresponding to 0.25 μg of thetotal protein digest) was separated on reverse-phase column(Ascentis Express Peptide ES-C18 150 × 0.2mm, 2.7 μm, 9 nm,Supelco) using Dionex UltiMate RP HPLC system. Peptides wereeluted by gradient starting from 2% to 50% B in 30min and ata flow rate of 1.5 μl/min (solvent A: 2% acetonitrile (ACN)/0.1%trifluoroacetic acid (TFA) and solvent B: 98% ACN/0.08% TFA).Eluted fractions were directly mixed with the loading buffer[2,5-dihydroxybenzoic acid (DHB) 20mg/ml in 50% ACN/0.1%TFA] and spotted onto a modified PAC using automated fractioncollector Proteineer fc II (Bruker Daltonics). Twenty-four minutesretention time window with time slice of 15 s resulted in 96fractions collected on the modified PAC MALDI plate.

J. Mass Spectrom. (2015) Copyright © 2015 John Wiley &

Preparation of Jurkat cell lysate for phosphopeptidesenrichment

Cells were seeded at 5× 105 cells/ml to final volume 50ml ofRPMI1640 medium and cultured for 24h in 150 cm2 cell cultureflasks under standard cultivation conditions. Phorbol 12-myristate13-acetate (PMA) was added at final concentration of 200 nM, andcells were treated for 15min. Cells treated with dimethyl sulfoxidewere used as control sample. Treated and control cells wereincubated for 15min in the hypotonic buffer with protease(Sigma-Aldrich) and phosphatase (Active Motif) inhibitors. After-wards, NP-40 was added at concentration of 1% (volume involume) to perforate cells and cell debris. Mitochondria and nucleiwere separated by centrifugation (14 000 g, 30 s). Proteins insupernatant were transferred to 50-mM ammonium bicarbonatebuffer using BioSpin columns (Bio-Rad), reduced by dithiothreitol(final concentration of 10mM in the sample), alkylated byiodoacetamide (final concentration 30mM in the sample) anddigested by trypsin overnight at 37 °C. Samples were then desalted,and the detergent was removed using the detergent removalprotocol described by Rey et al.[32] Dried samples were dissolvedin solvent A to final concentration of 10μg/μl, and volume corre-sponding to 50μg of the total protein digest was separated onMichrom PLRP-S 150×0.3mm, 5μm, 30-nm column (Michrom)and flow rate of 4μl/min. All other parameters were identical withthose used in the previously described procedure for the separa-tion of the model protein mixture. The sole exception was usageof a different loading buffer (for the cell experiments, DHB20mg/ml in 50% ACN/2% TFA).

In situ enrichment on modified MALDI plates

The PAC plate functionalized by ion landing and with 96 loadedHPLC elution fractions was placed into 80% ACN/0.1% TFA (80%ACN/2% TFA for Jurkat cells lysate) and washed for 2min. Afterdrying, 1μl of the 0.1% NH4OH (pH 11) was placed on each spot.Subsequently, 1μl of the DHB/phosphoric acid (PA) matrix(20mg/ml in 80% ACN/0.3% PA) was added to each spot and letto crystallize at room temperature. The peptide calibration standardmixture (Bruker Daltonics) wasmixedwith the DHB/PAmatrix (1 : 9),and 0.3μl of this mixture was deposited onto the 24 calibrationspots by a pipette.

Enrichment on TiO2 packed tips

Digested samples of either the standard model mixture or theJurkat cell lysates were enriched on the in-house packed TiO2 tipsaccording to the protocol described in the literature.[33] In brief,20μl of the model mixture (total protein concentration of0.05mg/ml) was loaded onto TiO2 beads (Titansphere 5μm, GLScience) packed in a GELoader tip (Eppendorf), and the protocolwas followed exactly as described in Thingholm et al.[33] The onlydeviation was the desalting step, which was performed onMicroTrap columns. For standard mixture, sample dried afterenrichment was dissolved in 20μl of solvent A. Fifty microgramsof proteins from Jurkat cell lysates were loaded on the packedTiO2 tip. Eluates from four tips were pooled, desalted, dried and dis-solved in 20μl of solvent A. Enriched mixture of phosphopeptideswas subsequently separated by LC as described before, mixed withthe DHB matrix and spotted on the standard AnchorChip plate(Bruker Daltonics). Instrument was externally calibrated usingpeptide calibration standard mix (Bruker Daltonics).

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MS and data processing

Mass spectra were acquired using the Ultraflex III MALDI TOF-TOFmass spectrometer (Bruker Daltonics) equipped with SmartBeam I(200Hz). MS spectra were collected in reflectron mode over theset mass range of m/z=700–4000. Tandem MS/MS spectra werecollected using the default LIFT method. A maximum of tenMS/MS spectra were acquired from a single spot. Measured datawere processed using FlexAnalysis 3.3 and BioTools 3.2 software(Bruker Daltonics) and searched by MASCOT (Matrix Science)against SwissProt database with a mass tolerance of 200ppm forMS and 0.8Da for MS/MS. The ion-score cutoff was 15 at signifi-cance threshold p< 0.05. While no taxonomy restrictions wereforced for the model mixture, the Jurkat cell lysate samples weresearched within the human proteins only.

Results and discussion

Matrix optimization for automated acquisition

The matrix commonly used for the analysis of the phosphorylatedpeptides is DHB. Unfortunately, DHB matrix does not easily crystal-lize homogenously, and long crystal needles are typically formedon the rim of the sample spot, which causes severe difficulties forthe automated MALDI data acquisition. There are several methodson how to obtain more homogenous DHB matrix crystallizationand distribution. Some additives such as spermine[34] or glycerol[35]

have been proven successful, but minimizing the sample spot areaand faster crystallization are more suitable alternatives that wereused in the presented work. One of the advantages of ion landingtechnique for MALDI targets modification is that the size of themodified spot can be freely adjusted by the slit mask during theTiO2 deposition. The diameter selected here was minimized to2mm for the regular sample spot (96 deposited) and 1mm forcalibration standard spot (24 deposited). Faster crystallization canbe achieved by using higher concentration of the organic solvent

Figure 2. Effect of the varying acetonitrile content in the matrix solution on thsolution was deposited on the TiO2 spot prepared by ion landing and let to dry

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in the matrix solution. The effect of increasing amount of acetoni-trile in matrix solution was investigated to obtain optimal crystalli-zation. The test was performed at 25 °C using dried-dropletcrystallization on the deposited TiO2 spots. As shown in Fig. 2,depositing matrix from 50% ACN still resulted in long individualneedles, which could be missed by the laser shot during theautomated measurement. Deposition from 80% ACN resulted inlong but thin needles densely covering the whole sample spot.The DHB crystallization from 90% ACN was very rapid and pro-duced a rugged crystal layer, which can cause split peaks in aMALDI spectrum. Consequently, 80% ACN was further used forDHB matrix deposition in all experiments.

Kjellström and Jensen[36] reported positive effect of PA as aMALDImatrix additive on phosphopeptide ionization, and their pro-posed DHB/PA matrix (20mg/ml of DHB in 50% ACN/49% H2O/1%PA) has become largely used for MALDI MS of phosphopeptides. Adisadvantage of the DHB/PA matrix is the increased contaminationof the MALDI ion source by the matrix deposits. This problem ismuchmore evident duringMS/MS experiments, where the numberof performed laser shots is significantly higher (the matrix depositson the ion source after just a single LC-MALDI-MS/MS experimentare documented in the Supporting Information in Figure S0). Suchdeposits especially disrupt high-throughput analysis. Thus, theconcentration of PA should be kept at the lowest possible level,which still allows significant signal enhancement. Figure 3 showsthe optimization of PA amount in the DHB matrix. In thisexperiment, the mixture of digested α-casein and β-casein,ovalbumin and BSA was enriched on a single spot by our standardprotocol. DHB/PAmatrix was then applied, and the signal intensitiesof the phosphorylated peptides obtained for different PA concen-trations (0.1%, 0.3%, 0.5%, 0.7% and 1%) were compared. DHBmatrix with 0.1% TFA – instead of PA – was used as a referencesignal level. The comparison in Fig. 3 was focused on the peak thatcorresponds to the quadruply phosphorylated β-casein fragment atm/z=3122Da. The results in Fig. 3 show that the biggest enhance-ment is observed already when 0.1% PA is used; no signal at

e 2,5-dihydroxybenzoic acid (20 ng/ml) crystallization. One microliter of eachat room temperature.

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Figure 3. Effect of the amount of phosphoric acid (PA) in the matrix solution on the intensities of phosphorylated peptides in matrix-assisted laserdesorption/ionization spectra. Phosphopeptides were enriched from the model mixture by the single-spot in situ method (without liquid chromatographyseparation). TFA, trifluoroacetic acid.

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m/z=3122 was detected without PA (top panel in Fig. 3), while apeak appears at m/z=3122 when only 0.1% of PA is added. Therelative intensity of the peak at m/z=3122 further increased whenthe PA concentration increased to 0.3%, but it has not changedsignificantly when the PA concentration was increased again to0.5%, 0.7% (not shown in Fig. 3) and eventually to 1% PA. As a resultof the previously described optimization experiments, DHB/PAmatrix was adjusted to the final composition of 20-mg/ml DHB in80% ACN and 0.3% PA for all experiments.

Enrichment of a model mixture and LC method testing

In the previous proof-of-principle report, the enrichment perfor-mance on individual TiO2 spots was evaluated using tryptic digestsof casein and in vitro phosphorylated trehalase as chosen modelanalytes.[23] To make this new technique usable in high-throughsample phosphopeptide analysis, we now present an improvedworkflow, which is based on coupling with a standard HPLC system.Chromatographic separation not only improves the automation

J. Mass Spectrom. (2015) Copyright © 2015 John Wiley &

but also allows to work with protein mixtures as well as withproteins obtained directly from cell lysates.

A model mixture of digested α-casein and β-casein, ovalbuminand BSA was used for the evaluation of the in situ enrichmentcombined with LC-MALDI-MS/MS approach. Total amount of0.25μg was loaded on the C18 column, separated and automati-cally deposited on the prepared TiO2 spots array. The eluatedeposition on modified MALDI plate was followed by the enrich-ment step, matrix deposition and MALDI-MS data acquisition.

Results obtained by in situ enrichment on modified MALDI platewere compared with the non-enriched sample and also with asample that was enriched by TiO2 packed tips as described byThingholm et al.[33] The objective was to demonstrate that perfor-mance of the in situ phosphopeptide enrichment on modifiedMALDI plates is not only convenient but also in no way inferior tothe standard techniques that use packed columns or tips. Fromthe workflow perspective, the plates-based approach is muchfaster, easier to perform and thus more suitable for high-throughput analysis.

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Table 1. List of the phosphopeptides identified by MS/MS search inenriched and non-enriched samples. Successful identification is markedby “X” in the table.

Phosphopeptide Theoreticalm/z

Non-enriched

Tipenrichment

In situenrichment

α-S1 casein

106–119 1660.79 X X X

104–119 1951.95 X X X

103–119 2080.05 X X

α-S2 casein

138–149 1482.61 X

138–150 1594.71 X

138–150 1610.70 X

25–41 2093.88 X

25–41 2109.88 X

β-casein33–48 2061.83 X

1–25 3122.26 X

Ovalbumin

340–359 2088.91 X

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Figure 4 shows spectra obtained as an extracted sum of acquiredMS scans (after HPLC separation) for non-enriched (top), packed-tipenriched (middle) and in situ enriched (bottom) samples. Asexpected, the spectrum of the non-enriched sample contains alarge number of peaks, most of them non-phosphorylatedpeptides. The total of 22 peptides was identified by MS/MS searchwithout enrichment, of which eight peptides belonged to BSA, ninepeptides to α-S1 casein and four peptides to β-casein. Surprisingly,no peptides originating from ovalbumin were identified by MS/MS.List of all identified peptides can be found in the SupportingInformation in Table S1. The spectrum from in situ enrichedsample contained more peaks than the spectrum of the sampleenriched by using packed tips, particularly in the spectrum rangeabove m/z=2200. For the base peak at m/z=1951, the in situobtained spectra demonstrated a 2.7 times better signal-to-noisevalue (S/N=2708) compared with the sample enriched by packedtips (S/N=1018). Signal-to-noise ratio is especially important forsubsequent tandem MS/MS data acquisition. Table 1 summarizesthe phosphopeptides identified by the search among MS/MSobtained fragments; these phosphopeptides confirmed by MS/MSare alsomarkedwith an asterisk in the single scan spectrum in Fig. 4.Only two phosphopeptides from α-S1 casein were identified in thenon-enriched sample, and they are actually products of trypsinmiscleavage. Three additional phosphopeptides were identifiedin the sample enriched using the packed tip. The peak atm/z=2080 is another miscleavage product from α-S1 casein,while m/z=1594 and 2093 are two unique phosphopeptides orig-inating from α-S2 casein. No phosphopeptides were identified fromβ-casein or ovalbumin by enrichment on packed tips. In situenrichment on a MALDI plate found nine phosphopeptides in the

Figure 4. Sum of all MS scans of non-enriched (top), packed tip enriched (middchromatography–matrix-assisted laser desorption/ionization–MS analysis of tsubsequent MS/MS analysis are marked with an asterisk.

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mixture: three from S1 casein, three from α-S2 casein, two fromβ-casein and one from ovalbumin. The phosphopeptidesidentified from α-S1 casein and α-S2 casein by both enrichmenttechniques are identical (the mass difference reported in Table 1is caused by methionine oxidation). Only the method based onthe in situ enrichment on MALDI plate was able to identifyphosphopeptides from β-casein and ovalbumin. It is reasonableto argue that better signal-to-noise ratio in the first MS scan prior

le) and in situ enriched (bottom) sample. All spectra were obtained by liquidhe protein model mixture. Peaks identified as phosphopeptides by the

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to the tandem MS/MS is the main factor that contributed toadditional identifications achieved by the in situ enrichment.Because an identical sample was separated by the same LCmethod for both enrichment experiments, one can concludethat the difference is related to the nature of the enrichmenttechnique.

The importance of improved signal-to-noise ratio can be furtherdemonstrated on the example of β-casein phosphopeptide atm/z=3122. In Fig. 4, this peak is present in both summed spectraof non-enriched sample and of in situ enriched sample. However,no MS/MS identification of the 3122 peak was obtained for thenon-enriched sample. Figure 5 compares the MS/MS spectra ofthe phosphopeptide at m/z=3122 for both samples. For non-enriched sample, it shows almost a flat line spectrum, with onlytwo peaks (Fig. 5, top): parent mass and neutral loss of H3PO4

(�98), which indicates low number of parent ions for subsequentmass selection and fragmentation by the instrument’s LIFT cell.Contrary to that, the bottom spectrum in Fig. 5 shows the MS/MSscan from the in situ enriched sample, which contains a significantnumber of fragment peaks. All were easily assigned by thesearching software. Some of these fragments are still phosphory-lated (corresponding peaks are marked with an asterisk in Fig. 5,bottom), which indicates that the covalent attachment of thephospho group survived the collision event but progressive lossesof phospho groups are also shown in this spectrum.

Figure 5. Comparison of the parent ion intensities of β-casein phosphopeptideMS spectra of non-enriched (middle) and in situ enriched (bottom) β-caseinacquired by automated liquid chromatography–matrix-assisted laser desorpmarked with an asterisk. Identified b-series and y-series fragments are marked i

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No precursor ion atm/z=3122was found in the sample enrichedby packed tips, which we again attribute to insufficient elution. Thisphosphopeptide is phosphorylated on four serine residues, whichlikely results in extremely strong binding on the TiO2 beads usedin packed tips and columns. The problem with elution of themultiple phosphorylated peptides has been reported[11,37,38] andwas most likely observed in the presented experiments as well.

Because the in situ technique allows for all peptides to betransported onto the MALDI plate and inside the ionization source,it does not suffer the same disadvantage. Even multiple phosphor-ylated peptides can be desorbed and ionized with the assistance ofthe acidic matrix and sufficient laser energy.[23]

Enrichment of phosphopeptides from Jurkat cell lysate

Enrichment analysis of protein phosphorylation in a biologicallyrelevant sample – a cell lysate – is much more challenging becauseof the complexity of themixture, expected low stoichiometry of thephosphorylation and more complicated sample matrix. Weselected Jurkat cells, immortalized human T-lymphocytes, whichare commonly used to study cell signaling in many differentbiological contexts. The experimental objective was to detectchanges in phosphorylation before and after a known signalingpathway of Jurkat cells was activated.

atm/z= 3122 between non-enriched and in situ enriched samples (top). MS/phosphopeptide at m/z= 3122 from protein model mixture. Spectra weretion/ionization–MS/MS analysis. Fragments containing phosphoserine aren the primary amino acid sequence of the phosphopeptide.

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Figure 6. Comparison of overall phosphopeptides identified in treatedcells and control cells by the two different techniques (top) andcomparison of phosphopeptides identified in treated and control cells byeach technique individually.

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In published studies, the initial amount of the sample for thephosphorylation analysis varies from 100μg to several milligramsof the digested protein.[38–40] Because the previously describedexperiments with the model protein mixture demonstrated a goodlimit of detection, we selected only 50μg of cell lysate proteindigest as a starting sample amount for the experiments involvingJurkat cells.The experiment again utilized HPLC separation and automated

spotting on themodifiedMALDI plate. Chromatographic conditions,enrichment protocols, matrix and MS tuning setup were all identicalwith the previously described protocols for our model mixture, andonly the loading and/or washing buffers contained 2% TFA.Activation of Jurkat cells was achieved by PMA treatment, and

identified phosphorylations were compared with those identifiedin untreated control sample. The performance of the in situ enrich-ment on MALDI plate was compared with the performance ofpacked tips for treated and control cell sample (Table 2).Table 2 shows that on average, 35% of proteins identified by

using enrichment on MALDI modified target were phosphorylatedcompared with 19% ratio achieved by enrichment using the packedtips. This indicates better selectivity of the modified MALDI target.Packed tip enrichment identified a higher total number of all

proteins and peptides in treated cells, while the number ofphosphorylated proteins was almost identical for packed tip (18)and in situ enrichment (16). This again shows that enrichment capa-bility is higher on the MALDI plate than by using the packed tip.Most importantly, only the enrichment on MALDI plate identifieda phosphopeptide at m/z=2700 (ELEAIFGRPVVDGEEGEPHSISPR)that originated from the mitogen-activated protein kinase (MAPK),a member of the MAPK/extracellular signal-regulated kinase (ERK)signaling pathway, which was activated by the prior PMA treatment(details about all identifications in the Supporting Information inTable S2).The number of proteins/peptides identified in the control cells

was lower compared with that for the treated cells. The packedtip technique again identified a higher number of all peptides, butboth methods again identified a comparable number of phospho-proteins (in situ enrichment identified ten phosphoproteins, andenrichment on packed tips is seven). The identified phosphopro-teins were involved in normal cell functionality (tubulin, tubulin-binding proteins or heat shock proteins). Unlike the treated cellsample, no phosphoproteins related to activated MAPK/ERK signal-ing pathway were found (Supporting Information in Table S2).The total number of identified proteins in the cell lysate is

negatively influenced by the intentionally chosen insufficient HPLCseparation. A fast HPLC gradient resulted in poor resolution ofpeptides, and even after HPLC separation, the collected fractionswere still highly complex. The existence of mixed MS spectrahindered the number of identified peptides/proteins. At the same

Table 2. Performance of the in situ and packed tip enrichment method in th

Proteins

Phosphorylated Total

Treated – in situ 16 47

Treated – packed tip 18 91

Control – in situ 10 28

Control – packed tip 7 37

Ratio is calculated as the number of identified phosphorylated proteins/pept

wileyonlinelibrary.com/journal/jms Copyright © 20

time, the MS/MS measurement was limited to ten scans perposition. We are aware of the fact that these are not idealconditions for such a complex protein mixture but the HPLC andMS/MS methods were selected to demonstrate that if targeted,the entire analysis can be performed fast enough to allow forhigh-throughput applications. To obtain more identifications,longer gradients could be implemented to achieve better chro-matographic separation, and the number of TiO2 spots on themodified MALDI plate can be easily increased by using the samerobotic device (e.g. from 96 to 384).

Interesting observations also originated from comparison of thetwo enrichment techniques. Although both methods are basedon the interaction of phosphopeptides with TiO2, the overlapbetween in situ and packed tip-based techniques was very low(see Fig. 6 for summarized diagrams or the Supporting Informationin Table S2 for details). Only three phosphoproteins wereconcurrently identified in treated cells by both methods and onlytwo in control cells. The two enrichment techniques are clearlycomplementary, despite the fact that they are based on the samechemical composition of the stationary phase. The SupportingInformation in Table S3 shows the identified peptides together withtheir pI values (before phosphorylation). Comparison of pI valuesfor phosphopeptides identified by in situ enrichment on MALDIplates and those identified by the tip enrichment shows that thein situ technique has a strong preference for more acidic phos-phopeptides. These are more strongly bound on TiO2 stationaryphase and are thus harder to elute if column-based enrichmenttechniques are used. This is in accordance with literature reportsand observations that LC–electrospray ionization–MS methods are

e analysis of the control and treated Jurkat cells protein digest

Peptides

Ratio Phosphorylated Total Ratio

0.34 23 78 0.29

0.2 30 156 0.19

0.36 13 50 0.26

0.19 8 63 0.13

ides over the number of all identified proteins/peptides.

15 John Wiley & Sons, Ltd. J. Mass Spectrom. (2015)

Workflow for identification of phosphopeptides

Journal of

MASS SPECTROMETRY

more efficient for smaller singly phosphorylated peptides.[41–43] Thesame problem with strong retention was not observed for in situenrichment. We speculate that formation of matrix excited statesafter the absorption of the laser energy results in super acidity ofsome of the matrix molecules,[44] which facilitates easier releaseand ionization of acidic phosphorylated peptides in the in situenrichment method. Because the proteins involved in MAPK/ERKsignaling are more acidic, in situ technique was especially advanta-geous for elucidation of phosphorylations related to that specificpathway. But both approaches should be considered complemen-tary from the overall perspective.

Comparison between treated and control cells revealed severalphosphoproteins as a response to the PMA treatment activatingMAPK/ERK signaling.[45] In this notion, mitogen-activated proteinkinase kinase 2 that is directly involved in the MAPK/ERK signalingwas identified using the in situ enrichment method. We haveidentified phosphorylation of several translation initiation factors(e.g. EIF3 and EIF5B) using the in situ enrichment method. Thesefactors may represent novel targets of MAPK/ERK signaling, andtheir phosphorylation suggests reprogramming of protein transla-tion following MAPK/ERK activation in Jurkat cells. Regulation oftranslational machinery by MAPK/ERK signaling was recentlydocumented.[46] Rho guanine nucleotide exchange factor 2 repre-sents known target of MAPK/ERK[47] and recently was describedas MAPK signaling amplifier.[48] Rho guanine nucleotide exchangefactor 2 was also identified as phosphorylated in PMA activated cellsusing the in situ enrichment method. tRNA-methyltransferase(NSUN2) represents another protein identified by in situ enrichmentafter PMA treatment. NSUN2 promotes tRNA stability and proteinsynthesis,[49] and together with translation, initiation factors can reg-ulate specific translation in Jurkat cells following PMA treatment.Another phosphorylated protein identified by the in situ enrichmentfollowing PMA treatment was bifunctional glutamate/proline-tRNAligase (EPRS). EPRS mediates gene-specific translational silencingfollowing its phosphorylation.[50] Altogether, in situ enrichmentanalysis confirmed activation of MAPK/ERK signaling in Jurkat cellsfollowing PMA treatment and revealed protein translationalmachinery as a newpotential target of MAPK/ERK signaling pathway.

Casein kinase I was identified by the tip enrichment. However,this enzyme is not involved in the canonical MAPK/ERK signaling,but cross talk between these two pathways was recentlydescribed.[51] Stathmin and calnexin phosphopeptides were identi-fied in control as well as treated cells using both enrichmentmethods. G3BP1 and YBX1 phosphopeptides were identified incontrol as well as PMA treated cells using in situ method. Surpris-ingly, tip enrichment identified G3BP1 phosphopeptide in PMAtreated cells only and YBX1 phosphopeptide in control cells. Otherphosphoproteins identified by the tip enrichment play a role inseveral cellular processes without known link to MAPK/ERK path-way andmay represent novel targets of this pathway in Jurkat cells.

9

Conclusions

It was demonstrated that application of TiO2 functionalized MALDItargets for the in situ enrichment coupled to the LC-MALDI-MS/MSoffers fast analytical workflow for identification of phosphorylatedpeptides from peptide mixtures and cell lysate. TiO2 was depositedon PAC MALDI targets by the automated ion landing surfacemodification technique, which was previously demonstrated asadvantageous for MALDI target functionalization. The low cost ofthe conductive polymer targets compared with polished steel

J. Mass Spectrom. (2015) Copyright © 2015 John Wiley &

makes the PAC targets optimal choice for high-throughput analysis.The MALDI matrix composition was adjusted to improve crystalhomogeneity and to lower the MALDI ion source contamination.The performance of the in situ enrichment on MALDI plates wasdemonstrated on a model mixture of proteins and Jurkat cellslysate. Results obtained for protein mixture revealed better condi-tions for MS/MS analysis using in situ enrichment. In addition, MAPKkinase 2 phosphorylation was detected in PMA activated Jurkatcells lysate. An analogous phosphorylation was not identified incontrol non-activated cell sample. The overall number of identifiedphosphoproteins can be simply improved by the use of a highernumber of TiO2 spots enabling more efficient HPLC separation.The comparison of the phosphopeptides identified by the in situand packed tip enrichment suggests that parallel use of bothtechniques is complementary and would also result in a highernumber of identifications.

Acknowledgements

This work has been supported by the Institutional Research Conceptof the Institute of Microbiology (RVO61388971), specific universityresearch (MSMT No. 20/2014) and grants from the Ministry of Educa-tion Youth and Sports of the Czech Republic and European RegionalDevelopment Funds (CZ.1.07/2.3.00/20.0055, CZ.1.07/2.3.00/30.0003and CZ.1.05/1.1.00/02.0109), Charles University (project UNCE204025/2012), Grant Agency of Charles University (800413), CzechScience Foundation (P206/10/P018, P206/12/1150, 14–21095P and13-16565S) and AMVIS Czech Republic – US exchange program(LH13051). The authors thank Vaclav Kobliha (Institute of Microbiol-ogy, now retired) for building automated ambient ion landing appa-ratus and DrW. Tim Elam (UWAPL) for many consultations regardingreactive landing and surface science. Access to instrumental andother facilities was supported by EU (Operational Program Prague– Competitiveness project CZ.2.16/3.1.00/24023). M. V.’s researchwas in part supported by UW APL. K. V.’s research was supportedby Neuron Foundation.

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Supporting information

Additional supporting information may be found in the onlineversion of this article at the publisher’s web site.

15 John Wiley & Sons, Ltd. J. Mass Spectrom. (2015)