Amoresano A

Chiappetta G

De Rosa C

Marino G

Palmese A

AMORESANO, ANGELA

Archivio della ricerca - Università degli studi di Napoli Federico II

SHORT COMMUNICATIONNovel method to investigate protein carbonylationby iTRAQ strategyAngelo Palmese & Chiara De Rosa & Giovanni Chiappetta &Gennaro Marino & Angela AmoresanoReceived: 12 April 2012 /Revised: 20 July 2012 /Accepted: 2 August 2012 /Published online: 29 August 2012# Springer-Verlag 2012Abstract This paper reports a novel methodology for rela-tive quantitative analysis of carbonylation sites in proteinsby exploiting a new isobaric tag for relative and absolutequantitation (iTRAQ) derivative, iTRAQ hydrazide(iTRAQH), and the analytical power of linear ion trapinstruments (QqLIT). Because of its operational simplicity,avoiding time-consuming enrichment procedures, this newstrategy seems to be well suited for quantitative large-scaleproteomic profiling of carbonylation.Keywords Protein carbonylation . iTRAQ . QuantitativeproteomicsIntroductionCarbonylation of proteins is a common type of damageresulting from oxidative stress in living cells [1, 2]. It isinduced by a variety of reactive oxygen species (ROS) aswell as by-products of lipid oxidation [3, 4].Several proteomic methods for the identification of car-bonylated proteins have been reported. Six different classesof reagents used to target carbonylated peptides werereported in a recent review [5]. Among these, biotin hydra-zide, introduced by Regnier [6] in 2005, has been quitebroadly used to study oxidized proteins in yeast [7], rats[8], and humans [9].However, in the fast-progressing post-genomic age thereis a growing interest in quantitative proteomic studies [10].A number of methods, aimed both at the identification andquantification of oxidation sites in proteins, have recentlybeen described. The use of light and heavy deuterium la-beled Girard’s Reagent P (1-(2-hydrazino-2-oxoethyl)pyri-dinium chloride) was introduced by Mirzaei and Regnier[11]. Differently oxidized samples were reacted with GRP-d0 and GRP-d5 and analyzed, after an affinity enrichmentstep, by reversed-phase high-performance chromatography(RP-HPLC) and tandem mass spectrometry for peptideidentification. Carbonylated peptides appeared as doubletclusters of ions separated by 5 Da or multiples thereof inrelation to the number of carbonyls that were present in thepeptide [11].In a similar way relative quantification studies havebeen suggested to be carried out by light and heavy13C6-labeled-dinitrophenylhydrazine (DNPH). Carbony-lated peptides appear, this time, as doublet clusters ofions separated by 6 Da or multiples thereof [12].A carbonyl-specific reagent, O-ECAT (oxidation-depen-dent element coded affinity tags), which can be loaded withvarious rare earth metals thus allowing differential quantita-tion of oxidized samples, was designed by Lee et al. [13].The widely used biotin hydrazide proteomic approachhas been turned into quantitative analysis by labeling thetrypsin-digested peptides, arising from affinity-enrichedcarbonylated proteins, with isobaric tags for relative andabsolute quantitation (iTRAQ) reagent(s) [14, 15].A. Palmese : C. De Rosa :G. Marino :A. Amoresano (*)Dipartimento di Scienze Chimiche,Università di Napoli Federico II,via Cynthia 4,80126 Naples, Italye-mail: angamor@unina.itG. ChiappettaSpectrométrie de masse biologique et protéomique,USR3149 ESPCI ParisTech,10 Rue Vauquelin,75005 Paris, FranceAnal Bioanal Chem (2012) 404:1631–1635DOI 10.1007/s00216-012-6324-9All the above methods suffer from several drawbacksparticularly in terms of recovery and reproducibility becauseof the erratic yields of affinity purification steps which areinvolved in most of them. Furthermore, it is well known thatin the biotinylation approach, biotin tags reduce the ioniza-tion efficiency and alter the fragmentation pattern of themodified peptide causing a loss in signal intensities and inthe quality of MS/MS spectra.Our research group recently reported an innovativeRIGhT (Reporter Ion Generating Tag) approach for theselective analysis of protein carbonylation, involving dansyllabeling of carbonylation sites [16]. Unfortunately no iso-tope coded dansyl regents are commercially available todirectly extend the above procedure into a quantitativeone. We therefore sought to expand the chemistry of iTRAQreagents by synthesizing iTRAQ hydrazide (iTRAQH) inorder to set up a novel methodology for the selective label-ing and relative quantitative analysis of carbonyl groups inproteins. In other words we suggest to avoid the doublelabeling procedure and the use of an affinity step as reportedelsewhere [14, 15] and to directly label the carbonylatedsites by using isobaric reagents, which are more sensitivethan isotopic tags [17]. Furthermore the tremendousanalytical potential of precursor ion scan (PIS) analysisin combination with the high sensitivity of linear iontrap instruments can be exploited, thus avoiding theenrichment steps.ExperimentalMaterialsTrypsin, α-cyano-4-hydroxycinnamic acid, sodium hy-pochlorite, standard proteins (bovine ribonuclease A(RNase A), chicken egg lysozyme, horse myoglobin,horse carbonic anhydrase, bovine serum albumin), am-monium hydrogen carbonate (AMBIC), and hydrazinewere from Sigma (St. Louis, MO). Acetonitrile (ACN)and trifluoroacetic acid (TFA) were from Romil (Waterbeach,Cambridge, UK). iTRAQ molecules were from ABSciex(Foster City, California, USA).Preparation of standard oxidized RNase A (OX-RNase A)Lyophilized RNase A was dissolved in a 5 mM NaClOsolution to a final concentration of 0.5 mg/ml. Thereaction was carried out at 37 °C for 15 min. Carbony-lated proteins were desalted by size exclusion chroma-tography on a Sephadex G-25 M column (GEHealthcare). Protein elution was monitored at 220 and280 nm. Protein fractions were collected, lyophilized,and stored at −20 °C.iTRAQH synthesisiTRAQH was synthesized from iTRAQ 114 and 117(1 nmol/μl dissolved in ethanol) and hydrazine (50 nmol/μl in ethanol) (molar ratio 2:1). The reaction was carried outat room temperature for 6 h. The product was purified byRP-HPLC on an Agilent Zorbax C18 column (4.6 mm×150 mm) (Palo Alto, California) using a 10 % to 65 % lineargradient in 20 min from water to ACN and verified byMALDI mass spectrometry.Carbonyl labeling with iTRAQHOX-RNase A was denatured and reduced as previouslydescribed [16] and then dissolved in 50 mM AMBIC, pH8.0. Trypsin digestion was performed using an enzyme/substrate ratio of 1:50 w/w at 37 °C for 16 h. Peptidesamples were lyophilized and then dissolved in 100 μl of10 % H2O, 6.5 % of TFA, 83.5 % ACN. An equal volume ofa 5 mM iTRAQH solution in ACN was then added and thesolutions were allowed to react at 37 °C for 18 h.Mass spectrometryMALDI experiments were performed on a Voyager-DE STRMALDITOF mass spectrometer (Applied Biosystems, Fra-mingham, MA) equipped with a nitrogen laser (337 nm). A1-μl aliquot of the total mixture was mixed (1:1, v/v) withα-cyano-4-hydroxycinnamic acid 10 mg/ml in ACN/50 mMcitrate buffer, 70:30 v/v.NanoLC-MS/MS experiments were performed on a4000QTrap mass spectrometer (Applied Biosystems), aQqLIT instrument, coupled to a 1100 nanoHPLC system(Agilent Technologies). Peptide mixtures were loaded ontoan Agilent reversed-phase pre-column cartridge (Zorbax300 SB-C18, 5×75 μm, 3.5 μm) at 20 μl/min with solventA (0.1 % formic acid, 2 % ACN in water, loading time5 min). Peptides were then separated on an Agilentreversed-phase column (Zorbax 300 SB-C18, 150 mm×75 μm, 3.5 μm) at a flow rate of 300 nl/min using 0.1 %formic acid, 2 % ACN in water as solvent A and 0.1 %formic acid, 2 % water in ACN as solvent B. The elutionwas accomplished by a 7–50 % linear gradient of solvent Bin 50 min. A nano-ionspray source was used at 2 kV withliquid coupling, with a declustering potential of 50 eV, usingan uncoated silica tip (o.d. 150 μm, i.d. 20 μm, tip diameter10 μm) from New Objectives (Ringoes, NJ). Spectra acqui-sition was based on a survey precursor ion scan for m/z 114/117. The Q1 was scanned from 400 to 1,400 in 2 s with“low” resolution, and the precursor ions were fragmented inq2 using a linear gradient of collision energy from 30 to80 eV. Finally, Q3 was set to transmit only ions at m/z 114/117 with “unit” resolution. This scan mode was followed by1632 A. Palmese et al.an enhanced resolution experiment for the ions of interestand then by enhanced product ion (EPI) experiments for thethree most abundant species. MS2 spectra were acquiredusing the best collision energy calculated on the basisof m/z values and charge state (rolling collision energy),scanning the mass range from 100 to 1,400 m/z at4,000 amu/s [18].Results and discussionA novel iTRAQ derivative, namely iTRAQ hydrazide(iTRAQH) (Fig. 1a), was designed by simply reactingiTRAQ, as its N-hydroxysuccinimide activated ester[17], with an excess of hydrazine. It is worth notingthat the formation of the hydrazide derivative does notFig. 1 a Reaction scheme forthe synthesis of iTRAQH. bModification of α-aminoadipicsemialdehyde residues byiTRAQHFig. 2 MS/MS spectrum of the precursor ion at m/z 1,307.7 attributedto iTRAQ-modified oxidized peptide 1–10 of RNase A. The spectrumcontains all the expected fragments of y and b series and by manualinterpretation it was possible to reconstruct the entire sequence of thepeptide 1–10 of RNase A retaining the modified residue. The modifi-cation site on the lysine residue in position 7 was inferred. The lowmass region of the spectrum showed the presence of the signals ofiTRAQ reporter ions that could be used for quantitative analysisProtein carbonylation by iTRAQ labeling 1633alter the isobaric nature of iTRAQ reagents as shown inFig. 1.In order to set up proof-of-concept experiments weattempted to prepare standard carbonylated derivatives offive model proteins (bovine ribonuclease A, chicken egglysozyme, horse myoglobin, horse carbonic anhydrase, bo-vine serum albumin) by using reported oxidation protocols[19] with different oxidants (i.e., hypochlorite and/or Fe2+/EDTA/H2O2). We aimed to selectively oxidize specific res-idues, without undesired side reactions, and to introduceonly a small number of modifications, in a controlled way,to mimic the in vivo sub-stoichiometric conditions. Unfor-tunately, MALDI analysis of the resulting oxidized specieswas satisfactory only for RNase A and lysozyme. However,in the latter case, the generated carbonyl moiety occurred atthe N-terminus thus forming a cyclic imine [16] and render-ing the modified residue unavailable to react withhydrazine-derived reagents.In the case of myoglobin, carbonic anhydrase, and bovineserum albumin, MALDI analysis of samples, either directlyand/or after tryptic digestion, showed that the parent pro-teins were either oxidatively degraded and/or underwentside-chain modifications unrelated to the carbonyl formation(data not shown). This result was not surprising because ofthe complex pattern of reactions generated by radical spe-cies [17]. Therefore only carbonylated RNase A, prepared asdescribed in the Experimental section (OX-RNase A), wasused to set up the method.MALDI mapping of OX-RNase A led to the verifi-cation of about 95 % of protein sequence and showedthe occurrence of a single anomalous signal at m/z1,149.63, thus showing that the rest of the proteinremained unaffected. The m/z 1,149.63 signal wasassigned to the oxidized peptide 1–10, possibly suggest-ing the conversion of a single lysine residue to α-aminoadipic semialdehyde. Moreover, the presence inthe MALDI mapping spectrum of a signal at m/z1,150.61, assigned to the unreacted peptide 1–10 ofRNase A, showed that the oxidation reaction was sub-stoichiometric. We considered the latter result quite inFig. 3 Magnified sections ofMS/MS spectra of modifiedpeptide 1–10 of RNase A for1:1 (a) and 1:4 (b) mixtures.The signals of reporter ions ofiTRAQ can be used forquantitative analysis ofcarbonylated peptides1634 A. Palmese et al.line with our intent because both the presence andabsence of oxidized species in the preparation actuallymimic the sub-stoichiometric nature of the oxidativemodifications [3].To test the effectiveness of the procedure for relativequantitation of carbonylated residues, fixed amounts ofOX-RNase A, in the nanomolar range, were digested withtrypsin and reacted with either iTRAQH 114 or iTRAQH117; the two different peptide mixtures were then mixed in a1:1 and 1:4 ratio, respectively, and analyzed.MALDI mapping showed the occurrence of a signal at m/z1,307.7 (ΔM0+158 Da) assigned to the carbonylated peptide1–10 modified with iTRAQH resulting in the formation of ahydrazone moiety, thus confirming the presence of a realcarbonylation moiety in the 1–10 sequence of RNase A.Because of the isobaric nature of iTRAQ molecules(Fig. 1b), differentially labeled peptides were indistinguish-able by MS analysis. The iTRAQ reporter ions generatedfollowing fragmentation events allowed the quantitativeanalysis by using MS/MS analysis. The two peptide mix-tures were analyzed by LC-MS/MS experiments in precur-sor ion scan (PIS) mode using the ions at m/z 114 or 117(i.e., the iTRAQ reporter ions) as targets for precursorselection. By exploiting the capability of the PIS mode,one can avoid the need for any enrichment step prior toLC-MS/MS analysis.To gain information about the carbonylation site, a sec-ond stage of mass analysis is performed. The MS/MS spec-trum (Fig. 2) led to the reconstruction of the peptidesequence with y ions and b ions retaining the modifiedresidue, clearly showing that carbonylation occurs at thelevel of the lysine in position 7, whereas the lysine inposition 1 remains unmodified.Moreover the presence of the iTRAQ reporter ions in thelow mass region of the MS/MS spectrum allowed us toperform the relative quantitative analysis. Figure 3 shows amagnified section of an MS/MS spectrum for both mixtures,in which the signals of iTRAQ reporter ions are shown. Byreplicating these experiments it was possible to calculate anexperimental ratio of 1.07±0.03 and 3.97±0.17 for the 1:1and 1:4 mixtures, respectively. These preliminary data dem-onstrate the feasibility of this method for relative quantita-tive analysis of protein carbonylation.ConclusionsThis communication reports preliminary results aimed atsetting up a new methodology for the selective qualitativeand quantitative analysis of carbonylation sites in proteinsby exploiting a novel iTRAQ-derived reagent. The strategysuggested here takes substantial advantage of QqLIT instru-ments to select specific labeled peptides giving rise to diag-nostic MS2 product ions. Owing to its operational simplicitythis new strategy seems to be well suited for the (a) directidentification of modified residues avoiding any enrichmentstep and (b) relative quantitation of carbonylated residues indifferent cellular states up to 4 or 8, given the availability ofiTRAQ 4-plex and 8-plex reagent kits. The reagent de-scribed when used in combination with other isotopicallylabeled tags, such as TMT [20], can provide quantitativedata which take into account the change of proteinabundances in relation to the extent of carbonylation.We have outlined a simpler and more effective approachfor quantitative proteomics studies in the field of proteincarbonylation with respect to other methods so far suggested[11–15]. The use of iTRAQH on real samples is currentlyunder investigation by analyzing carbonylation in differentprotein yeast extracts under different oxidative stressconditions.References1. Shacter E (2000) Methods Enzymol 319:428–4362. 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Novel method to investigate protein carbonylation by iTRAQ strategy.

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