Identification of Cross-Linked Peptides byHigh-Resolution Precursor Ion Scan

Amadeu H. Iglesias, Luiz Fernando A. Santos, and Fabio C. Gozzo*

Institute of Chemistry, University of Campinas, and Instituto Nacional de Ciencia e Tecnologia de Bioanalitica,CP 6154, 13083-970 Campinas, Sao Paulo, Brazil

Chemical cross-linking coupled to mass spectrometryanalysis has become a realistic alternative to the study ofproteins structure and interactions, especially when thesesystems are not amenable to high-resolution techniquessuch as protein crystallography or nuclear magneticresonance. One of the main bottlenecks of this approachrelies on the detection of cross-linked peptides, as theyare usually present in substoichiometric amounts incomplex samples. It was shown that one of the mainfragmentation pathways of disuccinimidyl suberate (DSS)cross-linked peptides yields diagnostic ions, whose struc-ture is composed of a rearranged lysine side chain andthe spacer arm of the linker. In this report, we demon-strate the feasibility of detecting these modified peptidesbased on a precursor ion scan in a quadrupole time-of-flight (Q-TOF) instrument. It was shown that the fragmen-tation of nonmodified tryptic peptides hardly generatesions with the same nominal mass of the diagnostic ions,making the precursor ion scan very specific to N-hydrox-ysuccinimide (NHS)-based cross-linkers. Moreover, theexperimental setup is the same as in the case of a regularcross-linking experiment, not demanding any additionalexperimental steps that would increase sample handling.The results obtained with protein samples allowed us topropose an algorithm that could be implemented in asoftware to process data from cross-linking experimentsin an automated and high-throughput way.

Protein structure, although of vital importance to understandbiochemical processes, represents a major analytical challengeas the use of high-resolution techniques is restricted to a relativelysmall number of proteins. Large interest exists, therefore, for thedevelopment of additional techniques that can obtain structuralinformation of a large number of proteins and be convenientlyperformed. Recently, chemical cross-linking coupled to massspectrometry (MS3D) has become an attractive alternative tointerrogate protein structure and interactions when no high-resolution method is applicable to the system of interest.1-3

In MS3D experiments, a protein or protein complex issubjected to the cross-linking reaction followed by enzymatic

digestion and mass spectrometry analysis. The identification ofcross-linked peptides can be used to reveal several structuralfeatures of proteins, such as solvent accessibility, protein dynam-ics, and distance constraints as well as interacting partners andinteracting domains in protein complexes. As this technique isbased on mass spectrometric analysis for the identification ofcross-linked peptides, it inherits all the attractive features of massspectrometry, such as high sensitivity, fast analysis, and datainterpretation, as well as applicability to virtually any protein.

The main bottleneck of this methodology, however, relies onthe identification and correct assignment of cross-linked peptides,since these species are formed in substoichiometric amounts.1

Several strategies have been developed to circumvent this limita-tion, such as isotope-coded cross-linkers,4,5 reagents with affinitytags,6,7 digestion in a mixture of H2

16O and H218O,8,9 and cleavable

cross-linkers.10,11 All these methods, however, require additionalsample handling and in most of the cases additional steps to thecross-linking reaction itself, decreasing sample throughputsensitivity.

As chemical cross-linkers behave similarly to post-translationalmodifications (PTM), it would be desirable to apply MS-basedmethods traditionally used for PTM in order to aid in the detectionof those peptides. One of those methods, precursor ion scan (PIS)on a triple quadrupole instrument (and more recently on hybridtriple quadrupole linear ion trap), has been widely used in thestudy of phosphopeptides,12-14 site determination of aldehydesmodification,15 identification and differentiation of N- and O-linked

* To whom correspondence should be addressed. E-mail: fabio@iqm.unicamp.br.

(1) Back, J. W.; Jong, L.; Muijsers, A. O.; Koster, C. G. J. Mol. Biol. 2003,331, 303–313.

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Jr. Rapid Commun. Mass Spectrom. 2009, 23, 1719–1726.(7) Zhang, H.; Tang, X.; Munske, G. R.; Tolic, N.; Anderson, G. A.; Bruce, J. E.

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L. J.; Maarseveen, J. H.; Muijsers, A. O.; Koster, C. G.; Jong, L. ChemBio-Chem 2007, 8, 1281–1292.

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Anal. Chem. 2010, 82, 909–916

10.1021/ac902051q 2010 American Chemical Society 909Analytical Chemistry, Vol. 82, No. 3, February 1, 2010Published on Web 01/11/2010

oligosaccharides in glycoproteins,16 and detection of glycopep-tides17 and glycoproteins.18 In 2002, Bateman et al.19 proposedthe use of a quadrupole time-of-flight (Q-TOF) instrument on apseudo-PIS experiment in which, instead of scanning the quad-rupole, alternate MS spectra were acquired with and withoutfragmentation. In this experiment, the quadrupole operates in thewide-band mode, while fragmentation is induced by increasingthe collision energy. If the fragment ion of interest is formed, thena regular product ion scan could be acquired. This would capitalizeon the advantages of this equipment, i.e., high speed of spectraacquisition associated with good sensitivity, resolution, and massaccuracy in both MS and MS/MS modes. More recently,Niggeweg et al. improved this method making it compatible withchromatographic separation.20 PIS on a Q-TOF instrument hasbeen used so far in the identification and quantitation ofphosphopeptides,21,22 detection and differentiation of argininedimethylation,23 and on the detection of glycosylated peptides.24

PIS-based analysis depends on the presence of a reporter ionin the MS/MS spectra, i.e., a fragment that is formed in thecollision cell for a class of compounds. In a previous paper bySeebacher et al., it was reported there was the formation of anion of m/z 222 in experiments of peptides containing disuccinim-idyl suberate (DSS) as the cross-linker.25 Our group has exploredthe fragmentation of those species, demonstrating its formationpathway.26,27 This ion (Figure 1) is formed by one rearrangedlysine residue connected to the DSS moiety in its acylium form.

Moreover, we have also shown the formation of two other ions(m/z 239 and 305) which can also be used as reporter ions forDSS-containing peptides. Previous reports demonstrate the abilityof different cross-linkers to generate gas-phase fragments whichindicate the presence of this reagent in the structure of theselected ion. Back et al. proposed the use of a benzyl-derivatizeddisuccinimidyl ester as a cross-linker.28 Upon low-energy frag-mentation, this reagent yields a stable benzyl cation marker ionwhich could be used to detect the presence of cross-linked species.Despite its simplicity, this reagent was used only for modelpeptides, and its use in the case of proteins was not shown so far.Tang et al. developed a new set of cross-linkers derived from apeptide amidation reagent which presents labile bonds upondissociation.29 Upon dissociation, this protein interaction reporter(PIR) yields not only a reporter ion, indicating the presence ofthe cross-linker, but as well the two intact peptides. However,practical aspects such as the commercial unavailability and difficultsynthesis, large size of the reagents, and the need for MS3 forthe identification of the connected peptides have diminishedthe interest in such cross-linkers. More recently, Soderblomand Goshe developed a dissociative cross-linking reagent(collision-induced dissociative chemical cross-linking, CID-CXL)in which the spacer arm contains the labile dipeptide Asp-Pro.30

As in the previous case, not only are those reagents not easyto obtain but they also need MS3 spectra to confirm the natureof the peptides. Moreover, if at least one of the peptidescontains an Asp-Pro bond, the identification of the cross-linkedpeptides will be even more troublesome. Therefore, in thepresent work we evaluate the detection and identification ofcross-linked peptides with DSS, the most used commerciallyavailable cross-linker in MS3D experiments, by means of a PISexperiment.

EXPERIMENTAL SECTIONMaterials. DSS and sequencing-grade modified porcine pan-

creas trypsin were obtained from Pierce and Promega, respec-tively. Peptides AGAKGAERLVKAGVR (PX), Ac-ARKGCREVT-KNDLR (P1), Ac-ARGKWPREVKIHR (P2), and Ac-ARYTKD-LSQRAFKGMR (P3) were obtained from Proteimax (Sao Paulo,Brazil). Chicken egg lysozyme was obtained from Sigma-Aldrich.All other reagents were obtained from Sigma-Aldrich and Tediaand used without further purification.

Cross-Linking Reactions. Cross-linking reactions were per-formed as previously described.26,27 Briefly, peptides were incu-bated in phosphate buffer 50 mM pH 7.5 with DSS in a 1:50 ratiofor 2 h, followed by quenching with Tris buffer pH 7.6 and trypsindigestion for 3 h. Some of the samples were spiked with a bovinehemoglobin trypsin digestion standard (Waters Co.) in a 1:1 molarratio.

Collision Energy Determination. In order to determine theoptimal collision energy to the formation of reporter ions, MS/MS spectra were acquired for different DSS-containing peptidesvarying the collision energy. Spectra were averaged for 10 s.

(16) Carr, S. A.; Huddlestone, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183–196.(17) Huddlestone, M. J.; Bean, M. F.; Carr, S. A. Anal. Chem. 1993, 65, 877–

884.(18) Haynes, P. A.; Aebersold, R. Anal. Chem. 2000, 72, 5402–5410.(19) Bateman, R. H.; Carruthers, R.; Hoyes, J. B.; Jones, C.; Langridge, J. I.;

Millar, A.; Vissers, J. P. C. J. Am. Soc. Mass Spectrom. 2002, 13, 792–803.(20) Niggeweg, R.; Kocher, T.; Gentzel, M.; Buscaino, A.; Taipale, M.; Akhtar,

A.; Wilm, M. Proteomics 2006, 6, 41–53.(21) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem.

2001, 73, 1440–1448.(22) Paradela, A.; Albar, J. P. J. Proteome Res. 2008, 7, 1809–1818.(23) Rappsilber, J.; Friesen, W. J.; Paushkin, S.; Dreyfuss, G.; Mann, M. Anal.

Chem. 2003, 75, 3107–3114.(24) Frolov, A.; Hoffmann, P.; Hoffmann, R. J. Mass Spectrom. 2006, 41, 1459–

1469.(25) Seebacher, J.; Mallick, P.; Zhang, N.; Eddes, J. S.; Aebersold, R.; Gelb, M. H.

J. Proteome Res. 2006, 5, 2270–2282.(26) Iglesias, A. H.; Santos, L. F. A.; Gozzo, F. C. J. Am. Soc. Mass Spectrom.

2009, 20, 557–566.(27) Santos, L. F. A.; Iglesias, A. H.; Gozzo, F. C. J. Am. Soc. Mass Spectrom.

2010, submitted for publication.

(28) Back, J. W.; Hartog, A. F.; Dekker, H. L.; Muijsers, A. O.; Koning, L. J.;Jong, L. J. Am. Soc. Mass Spectrom. 2001, 12, 222–227.

(29) Tang, X.; Munske, G. R.; Siems, W. F.; Bruce, J. E. Anal. Chem. 2005, 77,311–318.

(30) Soderblom, E. J.; Goshe, M. B. Anal. Chem. 2006, 78, 8059–8068.

Figure 1. Structure of the diagnostic ions of m/z 222.1494, 239.1759,and 305.2229.

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Liquid Chromatography/Mass Spectrometry Analysis.LC/MS/MS analyses were performed in a Q-TOF Ultima (Waters,Milford) coupled online to a nanoAcquity ultraperformance liquidchromatography (UPLC) system. An amount of 1 pmol of eachpeptide was loaded and desalted on a 180 µm × 20 mm WatersSymmetry C18. After the desalting step, sample was directed toa 100 µm × 100 mm Waters BEH130 C18 column at a flow rate of1.1 µL/min. Mobile phases A and B consisted of 0.1% formic acid/water and 0.1% formic acid/acetonitrile, respectively. The gradientconditions used are as follows: 0 min with 10% of B, then it linearlyincreased to 40% B in 25 min, then it increased up to 70% B in 28min where it remained until 40 min and in the next minute it wasdecreased to 10% of B. Typical operating conditions of the massspectrometer in PIS experiments are as follows: 3.5 kV (capillaryvoltage), 100 V (cone voltage), 100 °C (source temperature), and10 eV/25-50 eV for the low and high collision energies. Anytimethere was a fragment of m/z 222.1, 239.1, or 305.2 ± 0.2 Da, aproduct ion scan was acquired of the five most intense ions inthe MS spectrum.

RESULTS AND DISCUSSIONCollision Energy Optimization. In previous works,26,27 the

study of fragmentation patterns of both intra- and intermolecularcross-linked peptides revealed a set of diagnostic ions presentwhenever the peptide is bound to cross-linkers (Figure 1). ForDSS, for example, fragment ions of m/z 222.1494 and 239.1759are observed whenever the DSS molecule is attached to a peptide,being it an intra- or intermolecular species as well as a dead-end.The ion of m/z 222.1494 corresponds to the loss of ammonia fromm/z 239.1759. The fragment of m/z 305.2229, on the other hand,is specific to species where two lysine side chains are connectedby DSS, that is, intra- or intermolecular cross-linked peptides. Thisset of diagnostic ions can, therefore, be used in PIS experimentsto identify cross-linked peptides in the presence of nonmodified,regular species present in protein digests. Another remarkablefeature of this set of ions is that no isobaric a, b, or y fragmentsexist for the 20 common amino acids. This selectivity, coupled tothe high mass accuracy of TOF analyzers in both MS and MS/MS modes, should confer high specificity for the identification ofcross-linked peptide.

In the previous reports on the fragmentation of cross-linkedpeptides, the relative intensity of these diagnostic ions dependedstrongly on the collision energy, so this parameter was optimizedusing model cross-linked peptides (Scheme 1).26,27

The intensity plots of the diagnostic ions as a function ofcollision energy for intra- and intermolecular species with 2 and3 charges were determinate (plots for m/z 222, 239, and 305 canbe found in Supplementary Figures 1-3, respectively, in theSupporting Information). Formation of m/z 222 is strongly favoriteat lower collision energy in the case of the N-terminus cross-link(55 eV × 30 eV, Supporting Information Supplementary Figure 1A).It is not possible, however, to attribute this difference on the typeof cross-linking, since these peptides have very different masses.When the precursor masses are similar, the collision energyapexes are very close, indicating that a common collision energycan be applied as a function of m/z, just like regular peptides.This trend is also true for 2+ and 3+ intermolecular species(Supporting Information Supplementary Figure 1, parts C and D,respectively) as well as for the two other marker ions of m/z 239

and 305 (Supporting Information Supplementary Figures 2 and3). It is also worth mentioning that the 2+ intramolecular speciesof m/z 449.2 do not yield fragment ion m/z 305 at any collisionenergy as expected, since in this case the lysine residue isconnected to the N-terminus of the peptide and not to anotherlysine. On the basis of these results, all other MS/MS spectrawere acquired using the collision energy proportional to peptidem/z, which varied from 25 to 50 eV.

PIS on Cross-Linked Samples. The first sample analyzedby PIS on the Q-TOF was PX (Figure 2). This peptide containsthree possible sites for cross-linking: two lysines and the freeN-terminus. As can be seen in the low-energy chromatogram ofthe DSS reaction product, the major species present are the twoK-K (inter) and the K-N-terminus (intra- and intermolecular)cross-linked peptides. Non-cross-linked digested peptides anddead-end (non) digested species account for the other peaksobserved.

The extracted ion current (XIC) for the diagnostic ions in thehigh-energy chromatograms are shown in Figure 3. The firstobservation is the higher number of species in the XIC for theions of m/z 222 and 239 when compared to 305. This is inagreement to the fact that m/z 305 is only generated from intra-or intermolecular cross-linked peptides, whereas m/z 222 and 239are generated from all species containing the cross-linker moiety.It can be also noticed in the chromatogram for m/z 222 ion thatthe only intense species corresponds to the intermolecular cross-linked peptide; the other low-intensity signals were due to thepresence of M + 1 from m/z 304 ions. The XIC for m/z 222 and239, on the other hand, presents several species in addition tothe peptides mentioned above. These ions were mainly generatedfrom dead-end species. Owing to the intrinsic complex nature ofthe cross-linking reaction, there are several possibilities of dead-end peptides in this type of experiment: (i) single dead-end trypticpeptide, (ii) single dead-end with a missed cleavage site, (iii)multiple dead-end (two or three sites) tryptic peptides, and (iv)multiple dead-ends with a missed cleavage site. This large numberof possibilities explains the large number of signals detected inthis experiment.

Scheme 1. (A) Schematic Representation of theDesigned Peptides, Followed by Reaction with DSSand Trypsin Digestion and (B) Intra- andIntermolecular Species Formed from SyntheticPeptides Used in This Studya

a “x” presents a variable number and type of amino acid exceptlysine and arginine.

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The superimposed chromatograms of m/z 222 and 239 (Support-ing Information Supplementary Figure 4) compare the intensity ofboth ions. In general, m/z 222 presents higher intensity then m/z239, indicating that the loss of ammonia from m/z 239 is favorablein the energetic conditions used. For some low-intensity precursors,however, m/z 239 presents a higher intensity, and in some cases

only one of the ions is generated, as in the case of precursors withretention times of 14.5 and 19.4 min. Therefore, simultaneousmonitoring of both ions is required for more comprehensive analysis.This approach is more advantageous in Q-TOF type instruments(compared to tandem quadrupoles) because the addition of anadditional monitoring channel does not lead to sensitivity loss.

Figure 2. Low-energy chromatogram for peptide PX after reaction with DSS and trypsin digestion.

Figure 3. Extracted ion current for the three diagnostic ions for sample PX after reaction with DSS and trypsin digestion: m/z 222 (A), 239 (B),and 305(C).

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The next step consisted of simulating a sample obtained inexperiments with protein complexes. In this case, we spiked thePX sample with an equimolar amount of trypsin-digested bovinehemoglobin. This protein is composed by four polypeptide chainswith a total molecular weight of approximately 65 kDa and wouldtherefore constitute a reasonable “chemical background” for ourexperiments. Comparison of the low-energy chromatograms ofhemoglobin and hemoglobin + PX (Supporting InformationSupplementary Figure 5) shows that PX peptides elute earlier andthe marker ions should be present mostly in the 10-15 min elutiontime range, whereas regular hemoglobin peptides are eluted laterand no marker ions are expected during this elution time.

Figure 4 presents the normalized XIC for the diagnostic ionsin the digest hemoglobin with and without PX peptides. It is clearlyevident that none of the three marker ions are present in thehemoglobin sample, showing an excellent selectivity for cross-linked peptides. For the hemoglobin + PX peptides sample, onthe other hand, all the three marker ions are present in the elutiontime range expected for the PX peptides (10-15 min). Besides,it can be noted that the XIC of the marker ions is exactly thesame as that of the pure PX peptides (Figure 3), showing thatmatrix effects can be almost neglected, especially when coupledto a high-resolution chromatographic system (UPLC in this case),causing no considerable intensity loss when compared to theexperiment of pure PX peptides.

A striking feature of these marker ions is the high selectivity.The XIC of m/z 222, 239, and 305 for digested hemoglobin withPX peptides extracted with a width of 0.02 and 1 Da (SupportingInformation Supplementary Figure 6) show no major differencesin the chromatogram but only a slightly higher background noisefor m/z 305. The same experiment performed with lysozymedisplayed the same behavior (data not shown). This makes thePIS method also applicable in low mass accuracy instruments,like quadrupoles and ion traps.

The PIS approach was applied to an experiment wherelysozyme was cross-linked with DSS. As in the previous case, thelow-energy chromatograms for the samples with and withoutcross-linker show a clear difference in complexity (data notshown). XIC for ions of m/z 222, 239, and 305 once againdemonstrate the high selectivity of this approach toward cross-linked species, since no detectable chromatographic peaks areobserved in the absence of DSS (Figure 5). This is even clearerwhen both diagnostic ions for the presence of DSS (m/z 222 and239) are analyzed simultaneously, which can be done in a TOFinstrument without sensitivity loss.

Regular precursor ion spectra were acquired in order toconfirm the sequence of the modified peptides. For example, asmall chromatographic peak for both ions with retention time of16.5 min (Figure 6A) can be seen. Figure 6B shows the low-energy

Figure 4. Extracted ion currents of the diagnostic ions (curves A, B, and C correspond, respectively, to m/z 222, 239, and 305) for samplescontaining hemoglobin trypsin digest without (lower lines) and with (upper lines) the addition of PX reacted with DSS and digested with trypsin.

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Figure 5. Extracted ion currents of the diagnostic ions (curves A, B, and C correspond, respectively, to m/z 222, 239, and 305) for lysozymedigested with trypsin, reacted (upper lines) or not (lower lines) with DSS.

Figure 6. (A) Extracted ion current for m/z 222.1 and 239.1 for a lysozyme sample modified by DSS. The gray rectangle corresponds to thechromatographic peak summed up to generate the MS spectrum shown in panel B. In panel C is shown the product ion scan for m/z 381.7, inwhich the inset (D) shows the presence of m/z 222.1 and 239.1 but not 305.2.

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MS spectrum for this retention time, and Figure 6C shows theproduct ion scan for m/z 381.7, a dead-end type peptide. Thefragmentation pattern of this peptide allows the identification ofthe sequence KVFGR, where the first residue is the site ofmodification. This peptide sequence corresponds to the N-terminus of the protein, and therefore the modified residue is thefirst amino acid in the protein structure. The crystallographicstructure of lysozyme (PDB ID 1W6Z) clearly shows that thisresidue is very solvent-exposed and therefore prone to reactionwith DSS.

As described above, the proposed approach for the detectionof cross-linked peptides is very simple, once it does not requireany additional experimental handling or reaction of the sample,making it automatable for high-throughput cross-linking experi-ments. The next step to make this even more user friendly wouldbe to create an automatic pipeline for data interpretation to beimplemented in software.

The idea presented above could also be applied to otherN-hydroxysuccinimide (NHS)-based cross-linkers. Indeed, weshowed that cross-linkers homologous to DSS (DSG and DSSeb,with spacer chains containing 5 and 10 C atoms) fragment in asimilar way to DSS, yielding diagnostic ions analogous to the onespresented above.27 In all cases, the mass of these ions isdetermined only by the rearranged side chain of the lysine residue(83.0735) and the mass of the spacer arm of the cross-linker(Figure 1). The same experiment performed with lysozyme andDSS was done also with DSSeb and DSG (Supporting InformationSupplementary Figures 7 and 8), and results were very similar:owing to the high resolution of the instrument, it was possible togenerate XIC for the analogous diagnostic ions with low noiselevels, accounting for the high intensity of the signals in the

chromatogram. Supporting Information Supplementary Figures7A and 8A illustrate the XIC for ions of m/z 250 and 180, whichare homologous to m/z 222 (obtained from the experiments withDSS). The same was observed for the other two marker ions forDSSeb (m/z 267 and 333, in Supporting Information Supplemen-tary Figure 7, parts B and C) and DSG (m/z 197 and 263, inSupporting Information Supplementary Figure 8, parts B and C),analogous to m/z 239 and 305 from DSS experiments.

As a result, a general software algorithm is proposed, in whichdiagnostic ion masses are calculated based on the cross-linkerspacing chain (Figure 7). Basically, this algorithm would evaluateevery product ion spectrum looking for the presence of thediagnostic ions m/z SC + 83 or + 100 (within a certain massaccuracy), where SC is the mass of the cross-linker spacing chain;in the case of absence of these ions, this spectrum is discardedonce it corresponds to a peptide without the cross-linker moiety.The next step is to evaluate the presence of m/z 305; its absencecan indicate (i) dead-end type peptide, (ii) cross-link between Kand N-terminus, or (iii) cross-link between K and another residue.In case m/z 305 is present, this ion corresponds to K-K cross-linked peptide and, therefore, corresponds to an intra- or inter-molecular specie. In order to differentiate between intra- and inter-cross-linked species, the presence of y1 ions is verified: if it hasboth m/z 147.1 (y1 from Lys residue) and 175.1 (y1 from Argresidue), it means that this ion has two C-termini, i.e., anintermolecular cross-link. If it presents only one of this ions itcan be (i) intramolecular peptide, (ii) intermolecular peptide inwhich both chains have the same C-terminus, or (iii) intermo-lecular peptide in which one of the cross-linked peptides is theC-terminus of the protein and therefore its last residue can bedifferent from K and R. After this filtering step, the software would

Figure 7. General flowchart showing the algorithm for DSS homologous cross-linkers. SC represents the mass of the spacer arm chain of thereagent.

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interpret product ion spectra in order to try to get the sequenceof the linked peptide(s), based on the fragmentation patternsproposed previously.26,27

CONCLUSIONIn this report an approach based on PIS on a Q-TOF instrument

to detect cross-linked peptides was demonstrated. As previouslyshown, DSS-linked species upon CID yield diagnostic ions whichcan be used in PIS experiments. In the case of DSS, we haveshown that these ions are especially suitable for this approach,once there are not any y, a, and b fragment ions with same mass,resulting in experiments with high specificity. We also haveproposed an algorithm for the automated analysis of product ionspectra of candidate cross-linked ions. In this approach, precursorions are filtered by the presence of the diagnostic ions in itsfragmentation spectra, which is thereafter interpreted accordingto the fragmentation patterns proposed in previous works. Themethod is demonstrated to work with other NHS-based cross-linkers as well as other instrument types, like quadrupoles andion traps. The PIS approach is simple, does not require any

additional sample handling step, and is very selective, having thepotential to overcome the problem of cross-linked peptide iden-tification, one of the greatest bottlenecks in MS3D.

ACKNOWLEDGMENTThe first two authors have contributed equally to the work.

This work was supported by the Sao Paulo Proteome Network(FAPESP 2004/14846-0 and FINEP 01.07.0290.00) and InstitutoNacional de Ciencia e Tecnologia de Bioanalitica (CNPq 573672/2008-3 and FAPESP 08/57805-2). The authors also acknowledgeother support from FAPESP (2007/55930-1), CAPES, and CNPq.

SUPPORTING INFORMATION AVAILABLEAdditional information as noted in text. This material is

available free of charge via the Internet at http://pubs.acs.org.

Received for review September 11, 2009. AcceptedDecember 8, 2009.

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