Identification of post-translationally modified proteins in proteome studies

Albert Sickmann1

Katrin Marcus1

Heike Schäfer1

Elke Butt-Dörje2

Stefan Lehr3

Armin Herkner3

Silke Suer4

Inke Bahr5

Helmut E. Meyer1, 6

1Proteinstrukturlabor,Institut für PhysiologischeChemie, Ruhr-UniversitätBochum, Germany

2Medizinische Universitätsklinikfür Klinische Biochemieund Pathobiochemie,Würzburg, Germany

3Klinik II und Poliklinik fürInnere Medizin am Zentrum fürMolekulare Medizin Köln,Köln, Germany

4Institut für PhysiologischeChemie, Ruhr-UniversitätBochum, Germany

5Schering AG,Berlin, Germany

6Prot@gen AG,Bochum, Germany

Identification of post-translationally modifiedproteins in proteome studies

Proteome studies are powerful tools to solve many different problems in metabolism,signal transduction, drug discovery, and other areas of interest in life sciences. Upto now, high-sensitive methods for protein identification after two-dimensional gelelectrophoresis using mass spectrometry are available. However, the identification ofpost-translational modifications after two-dimensional gel electrophoresis is still anunsolved problem. In this paper, we want to give several examples for the successfulidentification of post-translational modifications and point mutations.

Keywords: Protein phosphorylation / Two-dimensional gel electrophoresis / Post-translationalmodification / N-terminal acetylation / Point mutations EL 4409

1 Introduction

A proteome has been defined as the protein complementexpressed by the genome of an organism, tissue, or dif-ferenciated cell [1]. The study of proteomes promisesto fill the gap between genome sequence and cellularbehavior of the expressed cells [2–4]. Commonly, twoproteomes are compared by a substractive analysis [5]in which differences due to drug treatment [3], cultureconditions [6], genetic variations [7], or diseases [8] canbe observed. The result is a number of protein spotswhich are up- or downregulated in the proteome. Theidentification of proteins from two-dimensional gels bymass spectrometry (MS) is a well-described technique[4, 9]. Both combined, protein separation by 2-D PAGEand MS characterization makes proteome analysis an

Correspondence: Dr. Albert Sickmann, Institut für Physiolo-gische Chemie der Ruhr Universität Bochum, Proteinstruktur-labor Gebäude MA2/143,Universitätsstraße150,44780Bochum,GermanyE-mail:[email protected];[email protected]: +49-0-234–321 4554

important tool for drug discovery, bacterial cell line opti-mization, examination of pathways in metabolism, signaltransduction, and many other areas of interest.

However, only in some cases all signals derived from asingle 2-D gel spot can be explained. This is a commonsituation, due to the fact that often more than one proteinis present in a single 2-D gel spot, or the protein is post-translationally modified at one or a few residues. Post-translational modifications lead to a pI and/or mass shiftof the protein. Examples are: phosphorylation, sulfation,and N-terminal acetylation leading to a pI shift which canbe resolved by high resolution two-dimensional electro-phoresis, but an additional mass shift is not detectable.In contrast to this, ubiquitinylation of proteins causes amass and a pI shift in a 2-D gel. In this paper, we describeapproaches for the successful identification of post-translational modifications as phosphorylation, methio-nine adducts, acetylation, etc. Some of the modified pep-tides are detectable in a common mass fingerprint spec-trum, but especially phosphorylated peptides need a spe-cific isolation before analysis. Commonly less than 1% ofthe protein is phosphorylated.

2 Materials and methods

2.1 In-gel digestion

Gel pieces derived from 2-D SDS gels are washed for10 min in digestion buffer (10 mM NH4HCO3, pH 7.8) anddigestion buffer/acetonitrile (1:1 v/v). These steps arerepeated three times. A final washing step with acetoni-trile leads to a shrinking of the gel piece, which is reswol-len with 2 �L protease solution (trypsin at 0.05 �g/�L indigestion buffer). Digestion is performed overnight at37�C. Gel pieces derived from 1-D SDS gels are reducedand alkylated before digestion. The gel pieces are

Electrophoresis 2001, 22, 1669–1676 1669

WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 0173-0835/01/0905–1669 $17.50+.50/0

Proteomicsan

d2-DE

washed one time with digestion buffer and the proteinsare reduced by addition of just enough reducing solution(80 mM DTT, 0.5 mM guanidinium-HCl, 0.8 mM EDTA, 0.1 M

Tris-HCl, pH 8.2) to cover the gel and incubated at 37�Cfor 30 min, then the reduced proteins are alkylated byadjusting the solution to 40 mM iodoacetamide with 0.5 M

iodoaceteamide, 0.1 M Tris-HCl, pH 8.2, and incubated atroom temperature for 15 min in the dark. The reaction isstopped by adding �-mercaptoethanol to a final concen-tration of 1 mM. The digestion is done as described above.

2.2 Sample preparations

For MALDI-MS after in-gel digestion: 10 �L of digestionbuffer is added to the gel piece, followed by incubationfor 30 min in a sonication bath. The supernatant is col-lected and concentrated using ZipTip pipette tips (Milli-pore, Bedford, CA, USA) according to the manufacturer’sinstructions. The samples are mixed on the target with asaturated matrix solution of �-cyano-4-hydroxycinnamicacid (Sigma, Deisenhofen, Germany) in 0.1% TFA/MeCN(1:1 v/v). For HPLC separation: 10 �L of digestion bufferis added to the gel piece and sonicated for 30 min. Thesupernatant with the extracted peptides is directly injectedinto the �-HPLC sample loop. The peptide extraction isrepeated twice. For MALDI-MS after �-HPLC separation:An aliquot (0.3 �L) of each fraction is mixed on the targetwith the same volume of a saturated solution of �-cyano-4-hydroxycinnamic acid (Sigma) in 0.1% TFA/MeCN(1:1 v/v).

2.3 ESI-MS

LC-MS/MS spectra are recorded on a Finnigan LCQ(Finnigan Mat, Bremen, Germany) ion trap mass spectro-meter equipped with a custom-built nanoelectrospray ionsource. Tryptic peptides are separated by reversed-phasenano-HPLC on a 75 �m ID�250 mm PepMap column(LC Packings, Amsterdam, The Netherlands) using a pre-column split; the flow rate is adjusted to 0.2 �L/min. Theextracted peptides are online concentrated on a �-pre-column (0.3 mm ID�5 mm). The solvent system consistsof (A) 0.1% formic acid and (B) 0.1% formic acid/84%MeCN. The gradient is 5–50% solution B in 40 min and50–99% B in 15 min. The collision energy is set automati-cally depending on the mass of the parent ion. Trappingtime is set to 200 ms and the automatic gain control is on.

2.4 MALDI-MS

MALDI-MS is done using a Reflex-III equipped with aSCOUT 384 ion source (Bruker Daltonik, Bremen, Ger-many). The acceleration voltage is set to 20 kV, and thereflection voltage to 21.6 kV. PSD spectra are obtained

with a parent ion selection of �30 Da. The reflection vol-tage is reduced in fourteen steps from 21.6 to 0.65 kV.Data acquisition is done on a SUN Ultra using the XACQsoftware, Version 4.0.2. Post analysis data processing isdone using the XMASS software, Version 5.0.

3 Results and discussion

3.1 Protein acetylation

The two cotranslational processes, cleavage of N-termi-nal methionine residues and N-terminal acetylation, areby far the most common modifications, occurring on thevast majority of eukaryotic proteins [10]. N-terminallyacetylated proteins show a special behavior in analysisof their tryptic peptides with MS. The acetylated N-termi-nal peptide commonly shows a very high ionization rateas demonstrated in Fig. 1. The MALDI mass fingerprintunequivocally identifies mouse cyclophilin A. However,the base peak (2048.99 Da) in the mass spectrum cannotbe explained by any unmodified peptide derived from themouse cyclophilin A sequence. The ion is selected fora MALDI-PSD experiment and the generated fragmention spectrum identifies this peptide as the acetylated N-terminal peptide Ac-VNPTVFFDITADDEPLGR. The spec-trum contains only y-ions due to the fact that the N-termi-nal NH2-group cannot be protonated anymore.

3.2 Protein phosphorylation

Phosphorylation of amino acid residues in proteins playsa major role in biological systems. Phosphorylation/de-phosphorylation acts as a molecular switch, controllingthe protein activity in different pathways as in metabolism,signal transduction, cell division, etc. Therefore, localiza-tion of phosphoamino acids in proteins is an importanttask in protein analysis. Commonly, tryptic peptides ofthe isolated phosphoproteins are analyzed by MS. Dueto the fact that radioactive labelling of the incorporatedphosphate is easy done with 32P, the method of choice isa separation of the phosphopeptide containing mixturefollowed by high-sensitive MALDI-MS analysis of theradioactive fractions. The three O-phosphates show dif-ferent behavior during MS and fragmentation analysis.However, phosphothreonine and phosphoserine losetheir phosphate group after fragmentation (Figs. 5 and 6).Since phosphotyrosine is more stable, the phosphategroup remains on this amino acid. A current review aboutphosphoamino acid analysis is given by Sickmann andMeyer [11].

The major problem analyzing phosphorylations is the sig-nal suppression of phosphopeptides in a mixture. There-fore, a high-resolution separation of the mixture before

1670 A. Sickmann et al. Electrophoresis 2001, 22, 1669–1676

Figure 1. (A) MALDI mass fin-gerprint of a 2-D gel spot. Theprotein cyclophilin A is identifiedby the peptide signals. How-ever, the signal with m/z2048.99 Da is not explained byany unmodified peptide of the

protein. (B) MALDI-PSD spectrum of the peptide ion with m/z 2048.99 Da. Only y-ions are obtained due to the fact, thatthe acetylated N-terminus cannot be protonated anymore. The nearly complete y-ion series is shown in the spectrum.I-type ions for phenylalanine (F, 120 Da) and C-terminal arginine (y1, 175 Da) are detected.

analysis or during analysis is essential for a successfulidentification of phosphorylated peptides [12]. Figures 2and 3 show a 2-D HPLC separation of a tryptic Gab-1digest. In the first dimension, anion-exchange chromato-graphy is done and fractions of 0.5 mL volume arecollected (Fig. 2). Fractions containing radioactivity areselected for a �-HPLC separation. The �-HPLC rechro-matography of these fractions is shown in Fig. 3. Frac-tions containing radioactivity are marked with an asterisk.In Fig. 4 the MALDI-PSD spectrum of the phosphorylatedpeptide TASDTDSSYpCIPTAGMSPSR which is identifiedin fraction c 59 is shown. The b- and y-ion series allow to

Figure 2. Anion-exchange chromatography of in vitrophosphorylated human Grb2-associated binder-1 (Gab-1)protein. The peptides are separated on a Nucleogel SAX1000–8/46, 50 mm�4.6 mm (Macherey & Nagel, Düren,Germany). The HPLC flow rate is set to 0.5 mL/minand peptides are eluted beginning with 100% solvent A(20 mM ammonium acetate, pH 7.0) and 0% solvent B(0.5 M KH2PO4, pH 4.0). The amount of buffer B is increasedto 10% over a course of 40 min and from 10 to 50% overthe course of 75 min. Fractions of 0.5 mL are collected.Radioactive fractions are pooled according to the solidbars (marked under the HAC-signals).

Figure 3. Reversed-phase separation of the fractions con-taining radioactivity (Fig. 2). The peptides are separated ona C18 reversed-phase column (250 mm� 0.8 mm, 5 �M

particle size, 300 Å pore size) using the ABI 140 D solventdelivery system (Applied Biosystems, Foster City, CA,USA). The HPLC flow rate is adjusted to 16 �L/min. Aftersample application, elution started with 95% solvent A(0.1% TFA) and 5% solvent B (0.08% TFA, 84% v/vacetonitrile). The content of solution B is raised to 50%over the course of 90 min. Fractions containing radio-activity (asterisk) are subjected to MALDI-MS.

Electrophoresis 2001, 22, 1669–1676 Post-translationally modified proteins 1671

Figure 4. MALDI-PSD spec-trum of the phosphopeptideTASDTDSSYpCIPTAGMSPSR.The phosphorylated tyrosineresidue is located with b- andy-ions in the primary structureof the protein. Due to the factthat tyrosine phosphate is astable phosphoamino acid, noloss of H2PO4 or HPO3 is ob-served.

determine the structure of the peptide and to localize thephosphotyrosine residue in the primary structure of theprotein.

Phosphoserine and phosphothreonine can be identifieddue to the loss of phosphate which is only observed inthe reflector mode but not in the linear mode of a MALDI-MS instrument. However, fragment ion spectra from pep-tides containing these two O-phosphates show in mostcases very intensive ion signals at [M+H]+ –80 Da (loss ofHPO3

2–) and [M+H]+–98 Da (loss of H2PO4–) in the reflec-

tor mode [13] as demonstrated in Figs. 5 and 6. The gen-eration of dehydroalanine from phosphoserine and�-amino-butyric-acid from phosphothreonine is a com-mon behavior of these phosphoamino acids. This be-havior makes it sometimes impossible to localize thephosphoamino acid by ESI-MS/MS or MALDI-PSDexperiments.

If a 32P-labelling is not possible, the analytical strategyhas to be changed to LC-MS/MS analysis, e.g., nano-HPLC coupled to an ion trap mass spectrometer. In asingle LC-MS/MS analysis more than 200 different pep-tides can be identified within 1 h. With this method a fastidentification of post-translationally modified proteins ispossible. The identification of an in vivo phosphorylationsite of yeast long chain fatty acid CoA ligase 2 is demon-strated in Fig. 7. The phosphorylated and nonphosphory-lated peptides elute together and are selected for frag-mentation within 8 s.

3.3 Modification of methionine during 2-D PAGE

Methionine is easily oxidized during sample preparationprior to 2-D PAGE. Using the IPG method [14], a furthermodification is introduced in methionine containing pep-tides. After isoelectric focusing and reduction/alkylationof proteins in the IPG gel, methionine occurs at a 48 Da

Figure 5. (A) MALDI-MS spectra of a radioactive HPLCfraction from a tryptic digest of the in vitro autopho-sphorylated PI4K92h. The ion with m/z 1848.8 Da isselected for a MALDI-PSD experiment (see B). The addi-tional peaks in the reflector mode represent typical frag-ments of phosphothreonine resulting from the loss ofH2PO4 and HPO3. (B) MALDI-PSD fragment ion spectrumof the designated phosphopeptide SVENLPECmcGITpHEQR. Nearly complete b-ion (N-terminal fragmen-tation) and y-ion (C-terminal fragmentation) series arevisible and unequivocally identify phosphorylation ofThr423. The typical generation of additional fragment ionsthrough the loss of phosphate during analysis is observedby a –80 Da and –98 Da shift of b- and y-ions.


Figure 6. (A) MALDI-MS spectrum of a radioactive 2-DHPLC fraction from a tryptic digest of NOS-III in vitrophosphorylated by cGK II. In the first dimension, the pep-tides are separated using a �-anion-exchange HPLC col-umn (0.3 mm�150 mm, PL-SAX, 8 �m, 1000 Å; LC-Pack-ings). Peptides are eluted at a flow rate of 4 �L/min with alinear gradient from 95% solvent A (20 mM ammoniumacetate, pH 7.0) to 99% solvent B (0.5 M KH2PO4, 25%v/v acetonitrile, pH 4.5) over 55 min. Fractions containingradioactivity are further separated using a 180 �m�250 mm C18 column (5 �m, 1000 Å; LC-Packings) ata flow-rate of 1.5 �L/min. Radioactive fractions aresubjected to MALDI-MS. The ion with m/z 1686.7 Da isselected for the MALDI-PSD experiment. Signals with anarrow represent typical fragments of phosphoserineresulting from a loss of H2PO4 and HPO3

-. (B) MALDI-PSD fragment ion spectrum of the designated phos-phopeptide KESpSNTDSAGALGTLR. Nearly completey-ion (C-terminal fragmentation) series are visible andunequivocally identify phosphorylation of Ser. The typicalgeneration of dehydroalanine from phosphoserine througha loss of phosphate during anlysis is observed by a –98 Dashift of the y-ions containing the phosphorylated serineresidue.

lower mass than before. Studies with the syntheticpeptide YGGFMTSEK showed a loss of 48 Da locatedat methionine after incubation with iodoacetamide (IAA)[15]. An example is given in Fig. 8. The signal at m/z

Figure 7. (A) ESI-MS/MS spectrum of the unphosphory-lated peptide VGLEPLTLEDDVVTPTFK. Nearly completeb- and y-ion series are observed. (B) ESI-MS/MS spec-trum of the phosphorylated peptide VGLEPLTLEDDVVTpPTFK. Besides the b- and y-ions several phosphory-lated y-ions are observed.

1743.87 Da shows two corresponding signals at m/z1759.86 Da (methionine sulfoxide) and m/z 1695.88 Da(loss of 48 Da, according to CH3SH). A further signal withlower resolution results from a PSD fragment of the pep-tide with m/z 1759.86 Da as described by Schnölzer andLehmann [16].

An additional example is given in Fig. 9. The MALDI-PSDspectrum demonstrates the localization of a modifiedmethionine residue in the primary structure of the trypich-Ras peptide VKDSDDVPMVLVGNK derived from anin-gel digestion. The loss of 48 Da is easily located atthe methionine residue, because nearly complete b- andy-ion series are observed in the MALDI-PSD spectrum.


Figure 8. (A) MALDI mass fingerprint spectrum of a 2-D PAGE separated protein from human liquor.The interpretation of the mass spectrum identifies human prostaglandin D2H isomerase. The signal atm/z 2211.10 Da is a peptide derived from porcine trypsine and is used for internal calibration. (B) Themass range m/z 1690 Da to 1760 Da is displayed. The peptide TMLLQPAGSLGSYSYR occurs in fourdifferent signals. At m/z 1743.87 Da the unmodified peptide, at m/z 1759.86 Da the methionine sulf-oxide peptide, at 1701.84 Da the major PSD fragment of the oxidized peptide and at m/z 1695.88 Dathe peptide with a loss of 48 Da are shown.

Figure 9. MALDI-PSD spec-trum of modified VKDSDDVPMVLVGNK from a trypticdigest of human h-Ras. Theloss of 48 Da is easily locat-ed at the methionine residue.Nearly complete b- and y-ionseries are observed.

3.4 Point mutations

Point mutations are another kind of modification inthe primary structure of proteins. The analysis of pointmutations by MS is not a common task. The identifica-tion of a single point mutation in a peptide is only possi-ble by manual interpretation of a fragment ion spectrum

derived from the modified peptide. Therefore, we selectpeptide signals from peptide mass fingerprints whichcannot be explained by the sequence of the identifiedprotein and analyze them manually. An example of apoint mutation in the primary structure of the mousepterin-4-�-carbinolamine dehydratase is demonstratedin Fig. 10. The signal at 1117.21 Da was selected for


Table 1. Sequence analysis results of hGab-1 phosphopeptides

Amino acid sequence of identifiedphosphopeptides

[M+H]+a) AA inh Gab-1

AA Fractionb) 32P (%)c)

HGMNGFFQQQMIYpDSPPSR 2320.0 230–248 Y 242 b26, d16 1,8VSPSSTEADGELYpVFNTPSGTSSVETQMR 3156.4 273–301 Y 285 f29 1,7TASDTDSSYpCIPTAGMSPSR 2197.8 365–384 Y 373 b6 2,7NVLTVGSVSSEELDENYpPMNPNSPPR 3024.4 431–457 Y 447 h12, g19, c59 27,6QHSSSFTEPIQEA-NYpVPMTPGTFDFSSFGMQVPPPAHMGFR

4367.0 458–498 Y 472 a63, a66–67,f35–38

30,7

FPMSPRPDSVHSTTSSSDSHDSEE-NYpVPMNPNLSSEDPNLFGSNSLDGGSSPMIK

6186.6 594–648 Y 619 b26, e25–26,h24–26, i24

17,8

QVEYpLDLDLDSGK 1574.7 654–666 Y 657 j17–18 6,1SSGSGSSVADERVDYpVVVDQQK 2392.1 675–696 Y 689 d16 11,6

a) Monoisotopic mass of the single charged ionb) Fraction after anion-exchange chromatography (letters)/reversed-phase HPLC (numbers)c) Total amount of radioactivity incorporated into the phosphopeptides corresponds to 100%

Figure 10. (A) MALDI mass fingerprint of a 2-D PAGEseparated mouse protein. The protein pterin-4-�-carbino-lamine dehydratase is identified after interpretation of themass spectrum. Except m/z 1117.21 Da all signals in thespectrum are explained by the protein sequence. Thepeptide with m/z 1117.21 Da is selected for MALDI-PSDexperiment. (B) MALDI-PSD spectrum of the ion with m/z1117.21 Da. The sequence AVGWNEVEGR is identifiedby b- and y-ions. A very intensive I-type ion at m/z 159 Dacorresponding to W is observed in the spectrum.

fragmentation and the sequence AVGWNEVEGR wasidentified. However, the corresponding database entrancewas AVGWNELEGR (m/z 1131.2 Da) and due to the V Lexchange the measured peptide mass is reduced by14 Da.

4 Concluding remarks

The identification of post-translational modifications inthe primary structure of proteins is an important task inprotein analysis. The identification of several modifica-tions, as phosphorylation and acetylation, are demon-strated. As shown in Fig. 1, the very high ionization rateof N-terminal acetylated peptides is a first evidence forthis modification. The fragment ion spectra only con-tain y-ions because b-ions are not protonated anymoredepending on the uncharged N-terminus. Peptides witha methionine residue occur in four different signals fromthree peptide species in a mass fingerprint spectrum. Allthree peptide species (the unmodified peptide, the oxi-dized peptide and the peptide with a loss of 48 Da at themethionine residue) can be separated by �-HPLC meth-ods and analyzed separately, indicating that the modifica-tion occurred before MS analysis and is not an artificialeffect.

The analysis of phosphorylated peptides is a very diffi-cult analytical task. Often a phosphorylated protein isvisible in a single 2-D gel spot, but in most cases thedirect localization of the phosphorylated residue in theprimary structure of the protein is not possible. In somecases, the phosphorylation rate of the protein is veryhigh (e.g., �-casein) and a positive identification is possi-ble with the material from a single 2-D gel spot. Com-monly about 1% of the total protein amount is phos-phorylated and signals from such peptides are not


detectable in a MALDI mass fingerprint [17]. Therefore,the method of choice is the purification of the phos-phorylated protein, followed by digestion and HPLCseparation. Depending on the protein size a high-resolu-tion 2-D separation is necessary to isolate the phospho-peptides, due to the high signal suppression of phospho-peptides during MS.

In our examples four different proteins are analyzed. Theyeast long chain fatty acid CoA ligase 2 (83 kDa) andPI4K92h (92 kDa) are separated by 1-D HPLC prior toMS analysis. The incorporation of radioactive phosphateis determined between 0.5 and 1% in the case of thePI4K92h and 25 pmol of total protein is used. The NOS-III (130 kDa) and GST-Gab-1 (123 kDa) fusion protein areseparated by 2-D HPLC prior to MS analysis dependingon the size of the protein and some copurified contami-nations like HSP, DMak. The total phosphorylation ofthese two proteins is about 0.5–1% and two (NOS-III) oreight (Gab-1) different phosporylation sites are identified(Table 1). The amount of sample is between 50 pmol(NOS-III) and 1 nmol (Gab-1). The results show that asuccessful identification of the phosphorylation sites isonly possible with a high amount of protein which can-not be applied to 2-D PAGE. Therefore, the purificationof a phosphorylated protein prior MS analysis is oftennessessary.

This research was supported by grant BU 740/2-1 andSFB 394 of the Deutsche Forschungsgemeinschaft.

Received October 16, 2000

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Identification of post-translationally modified proteins in proteome studies