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Isotopically Coded Cleavable Cross-linker for Studying Protein-Protein Interaction and Protein Complexes

2005; Elsevier BV; Volume: 4; Issue: 8 Linguagem: Inglês

10.1074/mcp.t400016-mcp200

1535-9484

Evgeniy V. Petrotchenko, В. К. Ольховик, Christoph H. Borchers,

Enzyme Structure and Function

An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative “cross-linked peptides” (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes. An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative “cross-linked peptides” (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes. Large protein assemblies play a key role in many biological processes. To understand the function of these protein complexes, studies of their structural organization and the protein-protein interfaces are of major interest to modern molecular biology. The conventional approach for studying protein-protein interactions in protein complexes is to use binary binding assays with pairs of protein subunits from the complex. However, this technique is unable to detect protein-protein interactions that are stabilized in the complex by cooperative binding and becomes laborious for multicomponent protein complexes.Current technologies for studying protein-protein interaction interfaces on a structural and molecular level, such as NMR and x-ray crystallography, are limited in application because they are restricted by considerations of protein amount, purity, concentration, size, and homogeneity (1Li T. Investigation of protein-protein interactions by isotope-edited fourier transformed infrared spectroscopy.Spectroscopy (Amsterdam). 2004; 18: 397-406Google Scholar, 2Chen C.J. Rose J.P. Newton M.G. Liu Z.J. Wang B.C. Protein crystallography.in: Modern protein chemistry. CRC Press LLC, Boca Raton, FL2002: 7-36Google Scholar). Furthermore, these technologies are only suitable for studies in vitro. Prior knowledge of the complex is required, and it is only possible to study one protein complex per experiment.An alternative approach to determine the architecture of protein complexes is the combined method of cross-linking with MS and MS/MS analysis (reviewed by Sinz (3Sinz A. Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes.J. Mass Spectrom. 2003; 38: 1225-1237Google Scholar)). This method has several advantages for the study of multiprotein complexes. First, in contrast to binary interaction assays, which preferentially reveal strong, stable interactions, such as those with high binding affinity constants, the cross-linking approach uses intact, functional, native protein complexes and has the capability of detecting less stable interactions (i.e. thermodynamically weak or kinetically labile) involving subunits at the periphery of the complex. In fact, interactions at the surface of the complex surface are expected to be the most easily identified using this approach because these interaction interfaces are the most easily accessible to the cross-linker. Second, novel methods have improved the sensitivity of detection and identification of components of protein complexes that are present at very low levels when purified in native form from biological systems (4Wine R.N. Dial J.M. Tomer K.B. Borchers C.H. Identification of components of protein complexes using a fluorescent photo-cross-linker and mass spectrometry.Anal. Chem. 2002; 74: 1939-1945Google Scholar). Finally, this method can be used to identify the actual sites of interaction on the cross-linked proteins (5Petrotchenko E.V. Pedersen L.C. Borchers C.H. Tomer K.B. Negishi M. The dimerization motif of cytosolic sulfotransferases.FEBS Lett. 2001; 490: 39-43Google Scholar).The procedure involves cross-linking of the entire protein complex followed by proteolytic digestion of the complex and MS identification of cross-linked peptides. Normally, cross-links can be detected by comparison of the proteolytic peptides obtained from the cross-linked versus non-cross-linked samples (3Sinz A. Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes.J. Mass Spectrom. 2003; 38: 1225-1237Google Scholar). Although the detection of cross-links is practical for small protein complexes, it is difficult to detect the relatively small number of cross-linked peptides among the hundreds to thousands of unmodified peptides from larger protein complexes. This has become the major challenge for this method. To overcome this obstacle, stable isotopically labeled cross-linkers have been used that serve as a specific marker for cross-linked peptides by providing a characteristic pattern of ion signals in the mass spectrum when used in a known isotopic ratio (6Chen X. Chen Y.H. Anderson V.E. Protein cross-links: Universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry.Anal. Biochem. 1999; 273: 192-203Google Scholar, 7Mueller D.R. Schindler P. Towbin H. Wirth U. Voshol H. Hoving S. Steinmetz M.O. Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis.Anal. Chem. 2001; 73: 1927-1934Google Scholar, 8Pearson K.M. Pannell L.K. Fales H.M. Intramolecular cross-linking experiments on cytochrome c and ribonuclease a using an isotope multiplet method.Rapid Commun. Mass Spectrom. 2002; 16: 149-159Google Scholar, 9Taverner T. Hall N.E. O’Hair R.A.J. Simpson R.J. Characterization of an antagonist interleukin-6 dimer by stable isotope labelling, cross-linking and mass spectrometry.J. Biol. Chem. 2002; 277: 46487-46492Google Scholar, 10Trester-Zedlitz M. Kamada K. Burley S.K. Fenyoe D. Chait B.T. Muir T.W. A modular cross-linking approach for exploring protein interactions.J. Am. Chem. Soc. 2003; 125: 2416-2425Google Scholar). Alternatively, isotopic labels can be incorporated into peptides during enzymatic cleavage, resulting in doubled mass increments because of presence of two proteolytic cleavage sites per cross-linked peptides (11Back J.W. Notenboom V. de Koning L.J. Muijsers A.O. Sixma T.K. de Koster C.G. de Jong L. Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling.Anal. Chem. 2002; 74: 4417-4422Google Scholar, 12Huang B.X. Kim H.-Y. Dass C. Probing the three-dimensional structure of bovine serum albumin.J. Am. Soc. Mass Spectrom. 2004; 15: 1237-1247Google Scholar), e.g. proteolysis in 18O-water will lead to the incorporation of 4× 18O in cross-linked peptides compared with 2× 18O in non-cross-linked peptides.The identification of the cross-linked peptides can be performed by exact mass measurement of the cross-linked peptides. The high mass accuracy of Fourier transform ion cyclotron resonance mass spectrometers (13Marshall A.G. Hendrickson C.L. Jackson G.S. Fourier transform ion cyclotron resonance mass spectrometry: A primer.Mass Spectrom. Rev. 1998; 17: 1-35Google Scholar) makes it ideal for this purpose, and Fourier transform ion cyclotron resonance has been used to successfully identify cross-linked peptides in model protein complexes (14Dihazi G.H. Sinz A. Mapping low-resolution three-dimensional protein structures using chemical cross-linking and fourier transform ion-cyclotron resonance mass spectrometry.Rapid Commun. Mass Spectrom. 2003; 17: 2005-2014Google Scholar). However, the number of cross-linked peptides matching an observed mass increases rapidly as the complexity of the system increases. For larger protein complexes, hundreds of possible peptide combinations may match an observed mass, especially when missed cleavage sites and the somewhat broad chemical reactivity of cross-linking reagents are taken into account. This occurs even for measurements with mass accuracies of better than 1 ppm. Furthermore, for proteomics applications, when identities of the interacting proteins are not known, the assignment of cross-linked peptides exclusively by mass is not feasible.Additional informational leading to unambiguous identification of the cross-linked peptides can be provided by mass spectrometric sequencing. Because of the branched structure of cross-linked peptides, however, fragmentation of intact cross-linked peptides is not very extensive, yielding in less informative MS/MS spectra. In addition, interpretation of MS/MS spectra can become problematic because fragmentation of both peptides, as well as the cross-linker, can occur. All this makes the unambiguous identification of the cross-links difficult if not impossible. In this situation the use of cleavable cross-linkers (15Bennett K.L. Kussmann M. Bjork P. Godzwon M. Mikkelsen M. Sorensen P. Roepstorff P. Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping-a novel approach to assess intermolecular protein contacts.Protein Sci. 2000; 9: 1503-1518Google Scholar, 16Back J.W. Sanz M.A. De Jong L. De Koning L.J. Nijtmans L.G.J. De Koster C.G. Grivell L.A. Van Der Spek H. Muijsers A.O. A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry.Protein Sci. 2002; 11: 2471-2478Google Scholar) is helpful because the two previously cross-linked peptides can be released and individually sequenced by MS/MS (17Davidson W.S. Hilliard G.M. The spatial organization of apolipoprotein a-i on the edge of discoidal high density lipoprotein particles: A mass spectrometry study.J. Biol. Chem. 2003; 278: 27199-27207Google Scholar).In addition to the challenge of detection and identification of cross-linked peptides, side reactions that can occur during the cross-linking procedure can limit the applicability of this combined approach (18Wong S.S. Chemistry of protein conjugation and crosslinking. CRC Press, Boca Raton, FL1991Google Scholar, 19Hermanson G.T. Bioconjugate techniques. Academic Press, San Diego, CA1996Google Scholar). Besides the desired interprotein cross-linking, the cross-linking reaction can lead to intercomplex cross-linking as well as to cross-linking within the same protein or even within the same peptide (intraprotein/intrapeptide cross-links). Cross-linker can also be incorporated into the protein by reaction at only one end without actually connecting any residues. The resulting peptides have been termed end-capped peptides or “dead end” cross-links (for nomenclature of peptide products obtained after cross-linking, see (20Schilling B. Row R.H. Gibson B.W. Guo X. Young M.M. Ms2assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides.J. Am. Soc. Mass Spectrom. 2003; 14: 834-850Google Scholar)). Additionally, second order cross-linking, where cross-linked peptides contain more than one cross-linker, adds another level of complexity (20Schilling B. Row R.H. Gibson B.W. Guo X. Young M.M. Ms2assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides.J. Am. Soc. Mass Spectrom. 2003; 14: 834-850Google Scholar). Intercomplex cross-linking can be minimized by careful control of experimental conditions, or these complexes can be separated by chromatography (6Chen X. Chen Y.H. Anderson V.E. Protein cross-links: Universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry.Anal. Biochem. 1999; 273: 192-203Google Scholar, 3Sinz A. Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes.J. Mass Spectrom. 2003; 38: 1225-1237Google Scholar), in contrast to intraprotein cross-links and dead end cross-links. Because interpeptide cross-links provide the most structural information about the protein-protein interactions, a specific method for detection of these cross-links would increase the usefulness of this approach for the study of complexes.In the study described here, we have used a custom-synthesized cross-linker, D12-ethylene glycol bis(sulfosuccinimidylsuccinate) (D12-EGS), 1The abbreviations used are: D12-EGS, D12-ethylene glycol bis(sulfosuccinimidylsuccinate); HIV, human immunodeficiency virus; RT, reverse transcriptase. 1The abbreviations used are: D12-EGS, D12-ethylene glycol bis(sulfosuccinimidylsuccinate); HIV, human immunodeficiency virus; RT, reverse transcriptase. that is both isotopically coded and cleavable. This D12-EGS combines the advantages of both isotope labeling and cleavage options, thus facilitating detection and identification of interpeptide cross-links. This isotopically coded cross-linker allows the detection of cross-linked peptides even in a very complex peptide mixture when it is used in a known ratio with its non-isotopically labeled analogue. Furthermore, the cleavage of this cross-linker allows us to quickly distinguish between dead end, intracross-linked, and intercross-linked peptides. The peptides obtained after cleavage of the cross-linker are still isotopically labeled and present characteristic mass increments and ion doublets or multiplets in the mass spectrum that are specific to the different types of cross-links. These peptides can readily be detected by MS, assigned to a particular type of cross-link, and can then be selected for sequencing by MS/MS. Thus, the novel isotopically coded and cleavable cross-linker described in this study facilitates the determination of protein-protein interactions in protein complexes.EXPERIMENTAL PROCEDURESMaterials—The cross-linker sulfo-EGS was obtained from Pierce (21Pierce Biotechnology Applications handbook and catalog. Pierce, Rockford, IL2003Google Scholar), and its deuterated derivative, possessing 12 aliphatic deuterium atoms instead of hydrogens in the linker region, was synthesized essentially as described in the literature (22Abdella P.M. Smith P.K. Royer G.P. A new cleavable reagent for cross-linking and reversible immobilization of proteins.Biochem. Biophys. Res. Commun. 1979; 87: 734-742Google Scholar). Briefly, ethylene glycol-d4 (Isotec, Miamisburg, OH) was reacted with succinic anhydride-d4 (Isotec) to yield ethylene glycolyl disuccinate, which was then activated with sulfo-N-hydroxysuccinimide in the presence of N,N′-dicyclohexylcarbodiimide. The final product was characterized by mass spectrometry. Based on the mass spectrometric analysis of the isotopically coded sulfo-EGS, the deuterium isotope content for the substitution of all 12 hydrogen atoms is estimated to be greater than 90% (see the ion signal labeled with an asterisk in the inset of Fig. 2, which corresponds to the D11-isotopomer). In this paper, the non-deuterated cross-linker is denoted as H12-EGS, and its deuterated derivative is denoted as D12-EGS. Model peptides (N-terminally acetylated peptides with single lysine residue) were purchased from Bachem Bioscience (King of Prussia, PA) or were synthesized by the University of North Carolina Peptide Sequencing Facility (Chapel Hill, NC). HIV reverse transcriptase (HIV-RT) was purchased from Worthington Biochemical Corporation (Lakewood, NJ). Ribonuclease S (RNase S) and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).Cross-linking and Sample Preparation—A 1:1 mixture of H12-EGS:D12-EGS was used for all cross-linking reactions. Model peptides at a concentration of 1 mm were cross-linked with equimolar amounts of the cross-linker mixture for 30 min at 25 °C in 0.1 m triethylammonium bicarbonate buffer, pH 8.0. Reaction was stopped by purifying/desalting the mixture using C18 Zip-Tips (Millipore, Billerica, MA), and the eluted peptides were mixed with MALDI matrix solution, a saturated solution of α-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid, 50% acetonitrile, and applied to a MALDI target plate. Proteins were cross-linked with 0.5 mm H12-EGS/D12-EGS in PBS, pH 7.4 for 30 min at 25 °C. The excess of cross-linker was quenched by adding Tris-HCl reaching a final concentration and of 0.1 m and pH 7.4 and incubating for 30 min at room temperature. The reaction mixture was then digested with porcine trypsin (Sequence grade, Promega, Madison, WI). HIV-RT was digested at a 1:20 enzyme:protein ratio at 37 °C overnight. For the proteolysis of the RNase S, 100 μg of the protein was incubated with 50 μl of immobilized trypsin beads (Pierce) at 37 °C overnight. The proteolytic peptides obtained were separated by reversed-phase HPLC (HP1100, Agilent Technologies, Inc., Palo Alto, CA) on a Vydac 218TP54 (C18, 5 μ, 250 × 4.6 mm) column (The Separations Group, Hesperia, CA) using a 60-min linear gradient of 5% to 65% acetonitrile in 0.1% trifluoroacetic acid at 1 ml/min. Fractions (1 ml) were collected and lyophilized, reconstituted in 10 μl of 0.1% trifluoroacetic acid:50% acetonitrile, mixed with matrix solution, and applied onto a MALDI target plate. In parallel, 2-μl aliquots were treated with 2 μl of 1 m ammonium hydroxide from 2 h to overnight at 25 °C to cleave the cross-linked peptides. The cleavage reaction mixture was directly applied to the MALDI target plate without further purification, allowed to dry to remove the ammonia, and combined with matrix solution.Mass Spectrometry and Data Processing—Mass spectrometric analyses were performed either on a Bruker Reflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA) or on an Applied Biosystems MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Framingham, MA) operating in reflectron mode. All tandem mass spectrometric experiments were performed on the MALDI-TOF/TOF mass spectrometer using air as the collision gas at a medium pressure setting (4e-007 torr) and a laser intensity of 5400 ABI units (Nd-Yag laser, 355 nm wavelength, 3–7 ns pulse, >12 μJ pulse energy.) The MALDI-MS spectra were screened for the presence of ion signal doublets/multiplets 12.07 Da apart and/or for the occurrence of doublets 4.03/8.05 Da apart in spectra obtained after cleavage of the cross-linker. The screening procedure for doublets has been automated by the use of a software program that we developed. In most of the cases, both ions of the doublet were simultaneously selected for MS/MS analysis by MALDI-TOF/TOF. To assign the cross-linked peptides, the MS-Bridge program (Protein Prospector, MS Facility, University of California at San Francisco, (23ProteinProspector,prospector.%20Ucsf.%20Edu/Google Scholar)) and software developed in-house were used to predict all possible combinations of cross-linked peptide masses and the masses or their cleaved peptides. The fragment ion spectra were manually matched to the predicted peptide fragmentation generated by Protein Prospector (University of California at San Francisco). Analysis of the crystal structure of HIV-RT (Protein Data Bank accession code 1RTH) and RNase S (Protein Data Bank accession code 2RNS) was performed with the use of the program RasMol (24RasMol www.Umass.Edu/microbio/rasmol/Google Scholar).RESULTS AND DISCUSSIONProof-of-principle Experiment—We have synthesized an isotopically coded EGS derivative that has the same structure as commercially available sulfo-EGS (Pierce) with 12 hydrogen atoms (H12-EGS) substituted for deuterium atoms (D12-EGS) (Fig. 1A). Both forms of EGS should exhibit nearly identical chemical properties, including reactivity of the NHS ester groups with lysine side chains and cleavage of the cross-linker with hydroxylamine; however the difference in mass of 12.0753 Da results in a characteristic doublet isotope pattern when applied as a 1:1 mixture. HIV-RT, a stable heterodimeric complex of 51- and 66-kDa subunits, was used as model complex to evaluate the utility of the novel cross-linker D12-EGS for identifying cross-linked peptides and to develop the experimental procedures for the cross-linking (Fig. 1B). Cross-linking of HIV-RT was performed with a 1:1 ratio of H12-EGS and D12-EGS, and the SDS-PAGE analysis showed that under the experimental conditions used a quantitative yield of cross-linked complex was achieved.MALDI-MS analysis was carried out after tryptic digestion of the cross-linked HIV-RT and separation of the peptides by reversed-phase HPLC. Fig. 2A shows a MALDI-MS spectrum obtained from the analysis of a single HPLC fraction with signals at m/z 1662.618 and 1674.490, which are consistent with EGS-cross-linked peptides or an EGS-modified peptide because (i) the ratio of the two ion intensities is ∼1:1, reflecting the ratio of H12-EGS and D12-EGS used in the cross-linking reaction, and (ii) the mass difference of the ion signals is in good agreement with the expected mass difference of H12-EGS and D12-EGS. To facilitate the assignment and identification of cross-linked peptides, a mathematical matrix was calculated containing the masses of all possible combinations of cross-linked peptides from HIV-RT, including masses from single peptides modified by EGS and masses resulting from missed trypsin cleavage sites. Using a mass tolerance of ± 0.3 Da (our standard value when using external calibration on the Reflex III MALDI-TOF) a single mass matching m/z 1662.618 was found in this matrix, corresponding to HIV-RT tryptic peptides 559KVL561 and 453LGKAGYVTNR462. This assignment was confirmed directly by tandem mass spectrometric analysis using a MALDI-TOF/TOF instrument. Both cross-linked peptides (Fig. 2B) were unambiguously identified from the sequence information.Fig. 2Proof-of-principle, mass spectrometric analysis of HIV-RT cross-linked with H12-EGS/D12-EGS.A, MALDI-MS spectrum of a single HPLC fraction. Cross-linked HIV-RT was digested with trypsin, and the resulting peptides were fractionated on a C18 HPLC column. This MS spectrum depicted ion signals at m/z 1662.618 and 1674.690, which differ by 12.072 Da and exhibit 1:1 signal intensity with the marker indicating the presence of the EGS cross-linker mixture. Further indication of EGS incorporation is the presence of the ion signal marked with an asterisk, which is characteristic of D12-EGS. A mathematical matrix (inset) was generated based on the equation shown above it to find potential pairs of cross-linked HIV-RT peptides that match EGS-specific ions. MW(EGS), molecular mass of EGS linker region as a free acid (262.0689 Da). Using this matrix, the ion signal at m/z 1662.618 can be assigned to the circled peptides. B, the MALDI-TOF/TOF analysis confirmed unambiguously the assignment of m/z 1674.690 to the [M(D12)+H]+ of the HIV-RT D12-EGS cross-linked peptides 559KVL561/453LGKAGYVTNR462. Sequence-specific y- and b-ions of the cross-linked peptides in the MALDI-MS/MS spectrum of [M(D12)+H]+ are labeled.View Large Image Figure ViewerDownload (PPT)By analyzing the crystal structure of the HIV transcriptase, we have determined a distance of 14.6 Å between the two cross-linked lysine residues, 559K and 455K (data not shown). This separation is within the length of the linker region of the cross-linker (16.1 Å) (21), demonstrating that under our experimental conditions, this H12-EGS/D12-EGS cross-linker mixture is suitable for determining protein-protein contacts in native protein complexes.Chemistry of Cleavage Reaction—Fig. 2B demonstrates that MS/MS sequencing of an intact cross-linked peptide is feasible. However, in most cases, non-informative and uninterpretable MS/MS spectra were obtained (data not shown) because of insufficient fragmentation. Therefore we investigated the conditions for cleavage of the cross-linker in intrapeptide, dead end, and interpeptide cross-links. In addition to an increase in fragmentation efficiency, cleavage of the cross-linker results in characteristic mass increments and characteristic doublet isotope patterns. These patterns are specific for the different types of cross-links because the peptides obtained after cleavage still contain part of the isotopically labeled cross-linker. Together with the observed mass increments to the uncleaved product, this allows one to easily distinguish between intrapeptide, dead end, and interpeptide cross-links.Cleavage of the ester bond of EGS under basic conditions should theoretically produce three sections of the cross-linker, the ethylene glycol moiety and two succinyl groups. Using model peptides we have found that cleavage generates cyclic N-succinimidyl moieties rather than free carboxyl groups (Scheme 1). To improve compatibility with the subsequent MALDI analysis, we used volatile ammonia solution rather than hydroxylamine as was originally suggested (25Swaim C.L. Smith J.B. Smith D.L. Unexpected products from the reaction of the synthetic cross-linker 3,3′-dithiobis(sulfosuccinimidyl propionate) dtssp with peptides.J. Am. Soc. Mass Spectrom. 2004; 15: 736-749Google Scholar). This eliminates the necessity for purification of the cleavage products thus preventing sample loss and increasing the sensitivity of the entire approach. As a result, the cleavage reaction mixture can directly applied on the MALDI target plate, dried to evaporate the ammonia, and reconstituted with matrix solution, which provides adequate peptide signal intensities in the MALDI-MS spectrum. With 0.5 m NH4OH solution and incubation overnight at 25 °C, the cleavage reaction is complete (data not shown). However, the use of shorter incubation times, leading to partial cleavage, has the advantage that both the starting and end products can be observed in the same MS spectra. This makes interpretation and assignment easier and less ambiguous, because more accurate mass differences between the intact and cleaved products can be obtained if they are in the same spectrum rather than being determined from separate experiments. Alternatively, cleaved and non-cleaved samples can be combined directly on the target plate.Scheme 1EGS cleavage mechanism and isotope pattern of cross-linked peptides in the mass spectrum of D12/H12-EGS cross-linked peptides. Cleavage of the D12/H12-EGS cross-linked peptides with 0.5 m ammonia causes the release of cross-linked peptides that contain isotopically coded N-succimidyl moieties. The peptides cross-linked with a 1:1 molar ratio of H12-EGS and D12-EGS show a 1:1 ratio of doublets 12.07 Da apart, whereas after cleavage of t