Quantification of Citrullination by Means of Skewed IsotopeDistribution Pattern

Marlies De Ceuleneer,†Katleen Van Steendam,

†Maarten, Dhaenens,

†Dirk Elewaut,

‡

and Dieter Deforce*,†

†Laboratory for Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium‡Department of Rheumatology, Ghent University Hospital, Ghent, Belgium

*S Supporting Information

ABSTRACT: Citrullination is a post-translational modificationof arginine, resulting in a loss of positive charge and a 1 Damass increase. Research on citrullinated proteins is crucial inrheumatoid arthritis, an autoimmune disease characterized bythe presence of antibodies against citrullinated proteins.However, little is known about the location or quantity ofdeiminated arginine residues in these proteins. Sincecitrullination gives rise to a mass gain of only 1 Da, theisotope pattern of the citrullinated and the noncitrullinatedversion of a peptide will overlap. However, the differencebetween the theoretical, or noncitrullinated, and the measuredisotope pattern can be used to quantify the amount ofcitrullination. We developed a method to quantify citrullinatedpeptides by means of their skewed isotopic distribution pattern. The method was first optimized with synthetic peptides, afterboth direct infusion and RP-HPLC separation on an ESi-QqTOF mass spectrometer. Additionally, we analyzed synovial fluidsamples from rheumatoid arthritis patients and were able to quantify citrullinated peptides originating from citrullinatedfibrinogen, a well-known antigen.

KEYWORDS: citrulline, rheumatoid arthritis, label-free quantitation, posttranslational modification

■ INTRODUCTION

Citrullination (or deimination) plays a role in manyphysiological processes, most notably apoptosis and epigeneticregulation, but also in pathologies such as cancer, multiplesclerosis, and rheumatoid arthritis.1 It is carried out by peptidylarginine deiminases (PADs), but this reaction might be self-regulating: PADs reach maximum citrullination efficiency 3 hafter their activation by calcium-influx, after which theyautocitrullinate.2 Due to this phenomenon, it is likely thatcitrullination of arginine residues is never complete. Therefore,it is of interest to investigate to which extent this posttransla-tional modification takes place.We chose to investigate citrullination in the context of

rheumatoid arthritis (RA). This autoimmune disease ischaracterized by the presence of antibodies against citrullinatedproteins (ACPA) in 70−75% of patients, with a specificity of95−99%.3 However, citrullination in itself is not specific for RA,and citrullinated proteins have been found in the synovial fluidof both RA and spondylarthropathy patients.4 The questionremains whether the nature of the antigen or the antigen loadmight play a part in the fact that some of these patients developantibodies against citrullinated antigens. In order to investigatewhether the extent of citrullination of a certain antigen plays arole in autoimmunity, the need arises for quantitative

techniques that can help determine a possible threshold fortriggering of autoimmune reactivity.Different problems arise in the quantification of citrulline-

containing peptides. Citrullination causes a 1 Da mass gain andthe loss of a positive charge, which changes the properties of acitrullinated peptide during LC-MS. The different interactionwith the RP-HPLC column can cause a shift in retention time,causing the citrullinated counterpart to elute at a later, butsometimes overlapping, time span. While it is possible to adjustthe LC-gradient to ensure complete separation of both peaks tofacilitate identification by MSMS, this would be time-consuming to optimize for all citrullinated peptides present ina sample and would be impractical for complex samples wherethe citrullination status might not always be known for allpeptides. Moreover, since citrullination in vivo is likely presentin very limited quantities,5 the complete separation will mostprobably result in a low ion count for the citrullinated peptide,hampering correct identification by tandem MS.Additionally, the loss of a positive charge will change the

charge state of the peptide, which means that the citrullinatedand noncitrullinated version of the same peptide will not

Received: May 16, 2012Published: September 17, 2012

Article

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© 2012 American Chemical Society 5245 dx.doi.org/10.1021/pr3004453 | J. Proteome Res. 2012, 11, 5245−5251

automatically share the same charge state distribution. So far,only one research group has been able to quantify in vivocitrullinated peptides from rheumatoid arthritis synovial tissue.5

They estimate the citrullination of two peptides fromfibrinogen-α (ESSSHHPGIAEFPSRGK and KREEAPSLR-PAPPPISGGGYRARPAK) between 1.2 and 2.5% by meansof label-free MS quantitation. However, they do not take intoaccount possible differences in charge state distribution due tocitrullination.Another attempt at quantification of citrullination was made

by Andrade et al.,2 who used iTRAQ-labeling to identifycitrullinated peptides in a mixture of autocitrullinated andnoncitrullinated PADs. While these authors mentioned thepossibility of using iTRAQ-labeling as a way to quantifycitrullination, they did not use it as such and therefore had lessneed of taking into account possible differences in charge statedistribution or overlap of MS spectra due to coelution ofcitrullinated and noncitrullinated peptides. Moreover, thismethod was carried out on artificial mixtures of noncitrullinatedand in vitro citrullinated PADs with the aim to identifycitrullinated residues in the citrullinated protein, and it has notbeen executed on in vivo samples.It is clear that there is a need for a straightforward method to

quantitate citrullination in in vivo samples. In this work, wepropose a practical way to estimate citrullination content fromqualitative data by means of the skewed isotope pattern of apartially citrullinated peptide. When a peptide is partiallycitrullinated, the isotopic distribution patterns of the citrulli-nated and the noncitrullinated PADs will overlap and cause adeviation from the expected isotopic distribution pattern. Weproved that this skewing in the isotope distribution pattern ofthe lowest charge state of a given peptide corresponds linearlywith the percentage of the citrullinated form of that peptidepresent in the sample. We also applied this method to synovialfluids of RA patients, in order to verify the usefulness of ourmethod for in vivo research.

■ EXPERIMENTAL SECTION

Peptides

All synthetic peptides were obtained from Thermo Scientificand were reconstituted in Milli-Q water (Millipore) and storedat −80 °C at a concentration of 10 mM.

Patient Samples

Synovial fluids were collected from patients after informedconsent, approved by the ethics committee of the GhentUniversity Hospital.

In Vitro Citrullination of Human Fibrinogen and CellLysates

In vitro citrullination was performed on human fibrinogenpurified from plasma (Calbiochem, Darmstadt, Germany) orHep-G2 cell lysates in deimination buffer (0.1 M trisHCl pH7.4, 100 mM CaCl2, 5 mM DTT), by adding 7 IU of PAD fromrabbit skeletal muscle (Sigma-Aldrich, St. Louis, MO, USA) permilligram of protein. After 2 h at 37 °C, the reaction wasstopped by adding EDTA to a final concentration of 50 mM.

Fractionation by Means of SDS-PAGE

When samples were too complex for direct infusion,fractionation was performed through gel electrophoresis. Forsynovial fluids, the sample was first treated with 1Uhyaluronidase (Sigma-Aldrich) per 10 μL of sample (30 min,37 °C). Afterward, the sample was dried and reconstituted in a

maximum of 1 mL of sample buffer (50 mM TrisHCl pH 6.8,2% SDS, 10% glycerol, 5% β-mercaptoethanol), incubated for 5min at 95 °C, loaded onto a 10% SDS-PAGE gel (CriterionTris-HCl gel, IPG+1lane, BioRad), and run for 30 min at 150 Vfollowed by 1 h at 200 V. After electrophoresis, a small part ofthe gel was used for Western blotting and the rest was cut into0.5 cm wide horizontal strips.

Western Blot and Immunodetection of Antigens

Proteins were transferred to a nitrocellulose membrane bymeans of transblotting with CAPS (50 V, 3 h). Afterward,vimentin and fibrinogen were detected by means ofimmunodetection with polyclonal antivimentin antibodies(Abcam Ab7114) and polyclonal antifibrinogen antibodies(Abcam Ab 6666). Briefly, membranes were blocked with 1%BSA/0.3% Tween in PBS and incubated overnight with primaryantibody diluted in blocking buffer (1/5000 for antivimentin,1/10000 for antifibrinogen). After incubation with secondaryantibody for 1 h (goat antirabbit, Pierce and rabbit antigoat,Abcam, respectively), bands were visualized by means ofSupersignal West Dura Extended Duration Substrate (Pierce).

Antimodified Citrulline Staining

For detection of citrullinated proteins on blot, the Anti-Modified Citrulline kit (Millipore) was used according to themanufacturer’s instructions.

In Gel Digest

Horizontal strips from the gel corresponding to the location offibrinogen and vimentin or other citrullinated proteins asdetermined by Western blot were subjected to in gel digestion.Selected bands were first washed three times in 250 mMTEABC (Sigma-Aldrich). After reduction with 25 mM TCEP(Sigma-Aldrich) for 2 h at 60 °C and alkylation with 200 mMMMTS (Sigma-Aldrich) for 1 h at room temperature, proteinswere digested overnight with 2 μg of chymotrypsin (Promega)at room temperature or GluC (Promega) at 37 °C. Afterward,extraction of the peptides was performed sequentially with 50%,75%, and 100% acetonitrile.

RP-LC

All separations prior to mass spectrometry were performed on aU3000 nano-HPLC device (Dionex) with a Pepmap C18column (15 cm, 5 μm particle size, 100 Å pore size, Dionex).Buffers were 0.1% formic acid (A) and 0.1% formic acid in 80%acetonitrile (B). Synthetic peptides were separated in a shortrun (4% B to 60% B in 15 min, followed by 5 min of 100% B).All other samples were separated over 70 min (4−100% B).

Mass Spectrometry Analysis

Data were acquired on a Waters Premier (ESI-QqTOF) massspectrometer after RP-LC separation. For tandem MS, collisioninduced dissociation was performed with custom collisionenergy profiles ranging from 25 eV to 55 eV for doubly chargedpeptides between m/z 400 and 1200 and ranging from 11 eV to26 eV for triply charged peptides between m/z 435 and 1000.

Identification of Peptides

Identification of peptides was performed by means of a MascotDistiller (Matrix Science, version 2.3.2.0). Peaks were pickedbetween m/z 50 and 100.000, with a correlation threshold setto 0.7 and a minimal signal-to-noise of 2. Baseline correctionwas applied for all spectra, and peaks were fitted by means ofisotope distribution. Mascot searches were all performedagainst the SwissProt database (version 57.15, humantaxonomy, 20 266 sequences) with methylthio on cysteine

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residues as fixed modification and deamidation of arginine,glutamine, and asparagine and oxidation of methionine asvariable modifications. The peptide mass window was 0.3 Da,and the window for fragment ions was 0.45 Da. The #13Cparameter was set to 0, to exclude the possibility that a peakcaused by partial citrullination was wrongly annotated as thesecond peak in an isotope cluster. All reported peptides weresignificant (p < 0.05) in Mascot’s protein family summary.

Calculation of Citrullination Percentages

For calculation of the amount of citrullination, the isotopicenvelope mixture model described by Dasari et al.6 wasimplemented in Excel using the Solver Add-In to determine theextent of deamidation (Supporting Information). First, thetheoretical isotope distribution is obtained from the “Isotopemodel” function in MassLynx and converted by the Exceltemplate into a graph with as many data points as theexperimental data based on hypothetical and user-definedintensity, mass error, and background noise values. Summedintensities are calculated for the noncitrullinated and thecitrullinated forms of the peptide separately, where the latterwill equal zero. The Solver Add-In, which optimizes thedifference between two data points, is then used to minimizethe intensity difference between the experimental and thetheoretical data by altering these intensity, mass error, andbackground parameters. From this new theoretical distribution,

the percentage of citrullination is calculated by dividing thesummed intensities of the citrullinated form with the summedintensities of all forms of the peptide.About 20% of deamidated and deiminated peptides showed

good chromatographic separation between the modified andthe nonmodified forms for the gradient described above. Mostof the peptides showed partial or complete overlap of thechromatographic peaks. When the modified and the non-modified versions of a peptide did not coelute completely, datawere obtained by integrating the extracted ion chromatogramsof both together.

Specific Modification of Citrullinated Peptides

Specific modification of citrullinated peptides in mixtures wascarried out by means of 2,3-butanedione (Sigma-Aldrich) in anacid environment as described previously.7

■ RESULTS AND DISCUSSION

Sample Preparation for the Analysis of CitrullinatedPeptides

A factor that needs to be taken into account when analyzingcitrullinated peptides is the amount of sodium ions in thesample. It became apparent that a citrullinated and anoncitrullinated version of the same peptide behaved differentlyin the presence of sodium and that the citrullinated peptide was

Figure 1. Calculated citrullination percentages for known mixtures of the citrullinated and noncitrullinated forms of synthetic peptides. (A) Theprocess of calculating the extent of citrullination by means of the Excel template. Dashed lines denote the theoretical isotopic distribution pattern forthe peptide STSRSLYASSPG (m/z 1212.5, [1+]); full lines denote the distribution pattern as measured in a sample containing 40% of thecitrullinated peptide. In the left panel, the theoretical distribution pattern is shown for the peptide without partial citrullination. On the right, thetheoretical distribution pattern is fitted to the measured distribution pattern, and from its alteration, the extent of citrullination is calculated. For thissample, containing 40% of STSCitSLYASSPG, the citrullination content was calculated to be 37.9%. (B) Calculated percentages of citrullination afterdirect infusion of samples containing increasing amounts of citrullinated peptide. Data are shown for all peptide charge states present in the samples.Trend lines were fitted by linear regression, and goodness-of-fit of the data to the calculated citrullination content is displayed (r2). For bothpeptides, the lowest charge state gives the highest correlation with the true citrullination content.

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more likely to form sodium-adducts (data not shown).Therefore, all samples were desalted for 30 min on a C18trapping column before nano-LC separation and subsequentMS analysis.Additionally, the possibility that citrullination altered

ionization capacity was investigated, since the loss of a positivecharge might diminish ionization capacity. For correctquantification based on peak areas, ionization of both peptidesneeded for the calculation should be comparable.8,9 Summedpeak intensities of all charge states of pure synthetic peptideswere comparable for the citrullinated and the noncitrullinatedform of the same peptide, showing that citrullination does notchange ionization capacity (data not shown).

Isotopic Distribution Pattern Is a Measure of CitrullinationContent of Peptides

The 1 Da mass gain introduced in a peptide by citrullination ofan arginine residue will alter the isotopic distribution pattern ofthis peptide when compared to its noncitrullinated counterpart.In the case of a complete enzymatic reaction, the spectrum willshift 1 Da but will retain the same isotopic distribution pattern,because the change in isotopic distribution introduced bysubstitution of a nitrogen and a hydrogen atom by an oxygenatom would be too small to be detected even by the mostaccurate mass spectrometers.10 However, when the citrullina-tion of the peptide is partial, the isotopic distribution patternsof the citrullinated and the noncitrullinated form of the peptidewill overlap. Since the presence in the environment of thedifferent isotopes of an element is known and fixed, the isotopicdistribution pattern of a peptide can be predicted with highconfidence. If the isotopic distribution of a peptide is altereddue to citrullination, the difference between the theoretical andthe measured isotope profile can be used to calculate theamount of citrullination.

Aside from the change in mass, citrullination also causes theloss of a positive charge. This has important consequences forthe charge of the peptide in an ESI source: the citrullinatedform of a peptide will have a lower charge state than thenoncitrullinated counterpart. This was investigated by means oftwo synthetic peptide pairs: SAVRARSSVTGVR/SAVRA-CitSSVTGVR and STSRSLYASSPG/STSCitSLYASSPG. Thecitrullinated and noncitrullinated peptide were mixed togetherwith the percentage of citrullination ranging from 0% to 100%,increasing in 10% increments. Data were obtained after directinfusion into the mass spectrometer and analyzed by means ofan excel template based on the isotopic envelope mixturemodel described by Dasari et al.6 In short, raw data are pastedinto the template, together with the theoretical isotopicdistribution of the peptide of interest, which is calculated bymeans of the “isotope model” function incorporated inMassLynx. By means of the Solver Add-in, the differencebetween both graphs is calculated and the amount of skewingof the isotopic pattern can be determined (shown in Figure 1Afor STSRSLYASSPG (1+) with a theoretical citrullinationcontent of 40%. The percentage of citrullination for this samplewas calculated to be 37.9%).When analyzing the different charge states of a peptide, we

could conclude that the lowest charge state of a given peptideshowed the highest agreement with the true percentage ofcitrullination (Figure 1B). For this lowest charge state, theslopes of linear regression were 1.072 (± 0.1035) forSAVRARSSVTGVR and 1 .003 (± 0 .03557) forSTSRSLYASSPG, indicating that the measured percentagewas a good gauge of the true amount of citrullination present ina sample. It is not surprising that higher charge states are morebiased toward the presence of arginine, since arginine can holda charge and citrulline cannot.

Figure 2. Optimization of the method after RP-nano-LC separation. (A) Comparison of low and high percentages of citrullination. Below 10%citrullination, no correlation can be found between the amount of citrullination present in the sample and the percentage calculated from the rawdata. When the sample is citrullinated for 10% or more, a good linear correlation is found. Goodness-of-fit of the experimental data to the calculatedcitrullination content is displayed (r2). (B) Calculated citrullination percentages for known mixtures of the citrullinated and noncitrullinated form ofsynthetic peptides, measured after separation by nano-HPLC (n = 2−3). Trend lines were fitted by linear regression.

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Optimization of the Method after LC Separation ofPeptides

The above data were collected by direct infusion of syntheticpeptides into the mass spectrometer, to provide proof-of-concept of the method. However, when analyzing real samples,the addition of an RP-LC step prior to mass spectrometryanalysis is preferred, to ensure maximal concentration ofindividual peptides. This results in a much lower sample loadneeded. Therefore, we expanded our method validation toinclude an LC separation step.First, we tried to evaluate the accuracy of our method when

looking at small and large amounts of citrullination. As can beseen in Figure 2A for the peptide SAVRARSSVTGVR, there isno linear correlation once the amount of citrullination dropsbelow 10%. Therefore, all calculated amounts that fall below10% will be displayed as “<10% citrullination”. This lowersensitivity shows that small differences in citrullination content,even at higher total citrullination contents, need to beinterpreted cautiously. The method as we describe here isless suitable for precise determination of the percentage ofcitrullination but rather should be used as an exploratorytechnique to compare the citrullination content betweensamples.Next, reproducibility of the method was assessed by analysis

of multiple dilution series of three peptide pairs: SAV-RARSSVTGVR and SAVRACitSSVTGVR, STSRSLYASSPGand STSCitSLYASSPG, and VNQDFTNRINKLKNSLFE andVNQDFTNCitINKLKNSLFE. The citrullinated peptides weremixed with their uncitrullinated counterparts from 0% to 100%in 10% increments, and this was repeated at least twice for eachpeptide to evaluate reproducibility (Figure 2B).The correlation of the measured and true percentage of

citrullination after separation on nano-HPLC was slightly lessthan with direct infusion, but the data still correlated linearlywith a good fit. The slopes of linear regression and goodness offit were 1.124 (± 0.0855) and 0.9234 for SAVRARSSVTGVR,1.064 (±0.083) and 0.9072 for VNQDFTNRINKLKNSLFE,and 0.933 (± 0.0627) and 0.9208 for STSRSLYASSPG. Thisconfirms that the observed extent of skewing of the isotopicdistribution pattern is a good measure for the actualcitrullination content in a sample. Across all samples theaverage deviation equaled just 4.4% (±10.1% SD). Thisstandard deviation emphasizes the fact that quantification oflow amounts of citrullination (<10%) is less indicated and thatthe method is more suited for the detection of larger differencesin the context of comparison of different samples.These data indicate that the method is usable for the

quantification of small sample amounts of complex mixturesafter RP-HPLC separation. However, slightly better correlationwas obtained after direct infusion, which should be the methodof choice if larger amounts of sample are available.

Citrullinated Loci in in Vitro Citrullinated Vimentin andFibrinogen

Citrullinated antigens of particular interest in RA are vimentinand fibrinogen, since both have been found in a citrullinatedform in synovial fluid.11 To investigate the usefulness of ourmethod in an in vivo setting, we chose to concentrate on theseproteins.First, we wanted to evaluate the feasibility to detect

citrullinated peptides from vimentin and fibrinogen in acomplex peptide mixture, such as a digest from a whole celllysate. To this end, 2 mg of a cell extract from Hep-G2 cells wasin vitro citrullinated and, after addition of 20 μg in vitrocitrullinated fibrinogen, separated by means of SDS-PAGE. Partof the gel was used for Western blotting, and after localizationof vimentin and fibrinogen, the corresponding gel bands weredigested and analyzed by means of LC-MSMS. Digests wereperformed with chymotrypsin and GluC in parallel. Amiscleavage of trypsin is often used for the identification ofcitrullinated peptides.12 However, C-terminal citrullination hasalso been reported elsewhere,13 indicating that miscleavageafter citrulline residues is not specific enough to be reliable.Additionally, in the case of partial citrullination and completeabolishment of trypsin specificity after citrulline, this wouldmean that the citrullinated and the noncitrullinated peptidewould no longer have a similar m/z, making the methodproposed here unusable.Two citrullinated peptides originating from vimentin (total

protein coverage 54.3%) and 1 from fibrinogen-α (total proteincoverage 14%) could be identified by Mascot (p < 0.05) andsubsequently quantified using our method (Table 1).For this analysis, we concentrated on peptides identified as

citrullinated by Mascot. However, this procedure is likely tomiss peptides that are citrullinated in small amounts. Thepeptide with sequence IATYRKLLE (VIME_HUMAN, expect-ancy 0.002) was annotated as unmodified, but after analysis ofits isotope profile, we could detect 26.5% skewing. This showsthat correct assignment of citrullination by Mascot will often beproblematic, especially for peptides with low citrullinationcontent. For future experiments we therefore investigated allpeptides containing internal arginine residues, which are allpotential citrullination sites.

Citrullinated Loci in in Vivo Citrullinated Fibrinogen fromSynovial Fluids of ACPA+ and ACPA− RA Patients

In RA, determination and quantification of citrullinatedepitopes might shed more light on the origin of theautoimmune reactivity. Synovial fluids from 24 RA patients,both ACPA+ and ACPA−, were screened on blot for thepresence of citrullinated proteins by means of AMC staining(data not shown). Four samples were selected based on theirstaining intensity. Two milligrams of each of these samples wasseparated on one-dimensional SDS-PAGE, which was then cutinto horizontal strips to provide a simple fractionation of thesample. Based on results from Western blotting withantivimentin and antifibrinogen antibodies, strips containing

Table 1. Three Citrullinated Peptides from Our Proteins of Interest Could Be Identified and Quantified

protein peptide sequence expectancy (p) m/z (charge) % skewing

VIME_HUMAN IKTVETRDGQVINETSQHHDDLE 0.00082 888.7 (3+) 63.1%

LNDRFANYIDKVRFLEa 1.8 × 10−005 672.05 (3+) 100%

FIBA_HUMAN TNTKESSSSHHPGIAEFPSRGKSSSY 0.00039 898.09 (3+) 100%aNo distinction could be made between citrullination and deamidation of asparagine, since both options were identified by Mascot.

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these known RA antigens were selected for in-gel digestion.Afterward, extracted peptides were analyzed by massspectrometry and the percentage of skewing of their isotopicdistribution pattern was calculated.No peptides from vimentin (citrullinated or other) could be

detected in any of the patient samples, even after use of anextensive include list. Likewise, no citrullinated peptides couldbe found for patient 3 (ACPA−). For all other patients (2ACPA+, 1 ACPA−), both the location of citrullinated epitopeson fibrinogen β and γ and the extent of citrullination werecomparable (Table 2).

Confirmation of Citrullination by Means of SpecificModification

For all peptides identified in the synovial fluid samples, we wereunable to distinguish between citrullination and deamidation ofasparagine (N) or glutamine (Q), since both modificationsresult in a 1 Da mass shift. Therefore, if both an arginine and anasparagine or glutamine residue are present in a peptide, thedata need to be interpreted with care, as has already been notedelsewhere.5 In order to confirm citrullination, we treated theremainder of the samples with 2,3-butanedione and TFAovernight, to specifically modify the citrullinated residues.7

This strategy was initially validated on six mixtures of thepeptides SAVRARSSVTGVR, SAVRACitSSVTGVR,VNQDFTNRINKLKNSLFE , and VNQDFTNCi -tINKLKNSLFE, prepared with increasing amounts of citrulli-nated peptide (0%, 20%, 40%, 60%, 80%, and 100%). Allsamples were modified overnight with 2,3-butanedione.Although the yield of the modification seems to be peptide-dependent, a drop in isotope skewing of at least 50% isapparent, and in most cases no residual citrullinated peptidescan be detected after modification (data not shown). Thisindicates the usefulness of an additional modification step toconfirm citrullination of the peptides.Subsequently, we also performed this confirmation step on

the synovial fluid samples. To this end, the remaining samplewas split in two and one part was modified by means of 2,3-butanedione in TFA, while the other part was incubatedovernight in TFA to serve as a control sample. Data-dependentmass spectrometry analysis of the control samples showedextensive deamidation of asparagine and glutamine as well asoxidation of methionine due to the long incubation in acid

environment. When analyzing the skewing of the isotopepatterns for peptides containing no arginines, we observed thatthis skewing was consistent across samples (data not shown).Therefore, we concluded that any difference in the isotopepattern between the modified and the control sample would bea result of modification of an initially citrullinated peptide. Afteranalysis, only a few peptides could be confirmed as citrullinated,as can be seen in Table 3. For both peptides, a drop of at least50% could be detected in the skewing of the isotopedistribution pattern, indicating the disappearance of thecitrullinated peptide from the extracted ion chromatogramafter modification. Residual skewing is due to deamidation ofthe asparagine or glutamine residues present in the peptide.To date, few studies have achieved the detection of

citrullinated peptides in in vivo samples, mostly relying on amiscleavage of trypsin to identify the presence of a citrullineresidue.12,14 However, C-terminal citrullination is reported inother cases,13 indicating the need for careful interpretation ofthe data. By means of specific modification, unambiguousassignment of citrullination is possible. Our study describes thefirst instance of successful modification of in vivo samples,making both identification and quantification of citrullinationpossible. Two separate runs remain necessary for modificationand quantification, because treatment with TFA results in lowerionization capacity. This hampers correct quantification bymeans of the deisotope excel template because peaks withlower ion count appear jagged and difficult to interpret.The peptide that could be quantified was previously detected

after in vitro citrullination using recombinant human PAD2 andPAD4.15 However, neither in vivo presence nor immunologicrelevance has been investigated yet. We could quantify the samepeptide in an ACPA+ and an ACPA− patient in similaramounts (13.3% in the ACPA+ patient and 12.4% in theACPA− patient), suggesting few distinguishing properties, butin order to explore this notion further, a larger amount ofsamples should be investigated. This lies outside the scope ofthe current investigation, however, since the aim of this studywas predominantly the optimization of the quantificationmethod.

Table 2. Citrullinated Peptides Found in Three Synovial Fluid Samples from RA Patientsa

patient protein peptide sequence expectancy (p)a m/z (charge) % skewing

1 (ACPA+) FIBB_HUMAN RGTAGNALMDGASQL 0.0001 731.4 (2+) 50.7

ALLQQERPINRNSVDE (0.0002) 884.4 (2+) 13.3

FIBG_HUMAN QKRLDGSVDF 0.007 582.8 (2+) 31.2

SSKPNMIDAATLKSRKMLEE (1.8 × 10−10) 1124.6 (2+) 6.3 (<10%)

2 (ACPA+) FIBB_HUMAN RGTAGNALMDGASQL 0.031 731.4 (2+) 58.1

4 (ACPA−) FIBB_HUMAN TVIQNRQDGSVDF 0.00075 738.9 (2+) 15.9

ALLQQERPINRNSVDE (0.00049) 884.4 (2+) 12.4

RGTAGNALMDGASQL 0.023 731.4 (2+) 64.1aFor peptides where only the higher charge state was identified by Mascot, the lower charge state was inferred from the raw data by detection ofcoeluting peptides with the correct m/z. For these peptides, the expectancies of the higher charge states are shown between brackets. Digests wereperformed in parallel with GluC and chymotrypsin.

Table 3. Quantification of Peptides with a Confirmed Citrullinated Residue

patient peptide sequence m/z (charge) % skewing in control sample % skewing in modified sample

1 (ACPA+) ALLQQERPINRNSVDE 884.4 (2+) 100 49.5

4 (ACPA−) ALLQQERPINRNSVDE 884.4 (2+) 87.8 6.9

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■ CONCLUSIONS

In rheumatoid arthritis, antibodies against citrullinated proteinsplay a central role. While many of the putative citrullinatedantigens have been identified, little is known about the extent ofcitrullination. It is possible that a certain threshold should beexceeded before a citrullinated protein becomes antigenic, andtherefore, it would be useful to quantitate citrullination in vivo.We developed a method to quantify the extent of

citrullination based on the alteration of the isotopic distributionpattern of a peptide. Since no modifications of the sample orspecial equipment are needed, this approach can also be appliedto data that were acquired previously, which createsopportunities to retroactively quantify citrullination in patientsamples where before only identification was possible.However, in order to distinguish citrullination from deamida-tion of asparagine or glutamine, a modification step can beadded in a separate run.The quantitative method was evaluated on citrullinated

synthetic peptides, and a good linear correlation could be foundbetween the actual and the measured citrulline content of allsamples. Additionally, to prove its usefulness in an in vivosetting, citrullination was estimated in fibrinogen derived fromRA synovial fluid samples.Only one paper to date has reported quantification of in vivo

citrullination,5 with the percentage of citrullination detected insynovial tissue from four rheumatoid arthritis patients rangingbetween 1.2 and 2.5%. These data were obtained throughaccurate mass and retention time analysis, which is a time-consuming procedure due to the need for determination ofmass and retention time preceding analysis of the samples. Thepercentages found in our study are slightly higher, which mightbe due to the fact that a different matrix was investigated(synovial fluid versus synovial tissue). Since ACPA are highlypresent in synovial fluid of ACPA positive patients,16 it ispossible that citrullinated antigens are more concentrated inthis compartment. Additionally, a concentration of certainepitopes might happen in immune complexes, making them ofparticular interest to investigate the importance of citrullinatedresidues and their quantity.In conclusion, our method for quantification of citrullination

is useful in in vivo settings, and its application to a wider varietyof samples might shed a new light on the importance of theamount of post-translational modification in autoimmunity.

■ ASSOCIATED CONTENT

*S Supporting Information

Mascot peak tables and annotated spectra, and Excel templateworksheet for fitting isotopic distributions to mass spec data.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author

*E-mail: dieter.deforce@ugent.be.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported by a grant from the Fund of ScientificResearch Flanders (FWO). The authors would like to thank Dr

Phillip Wilmarth for the Excel template that was used forcalculation of the citrullination percentages.

■ REFERENCES

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Journal of Proteome Research Article

dx.doi.org/10.1021/pr3004453 | J. Proteome Res. 2012, 11, 5245−52515251