An Isotopically Coded CID-cleavable Biotinylated Cross-linker for Structural Proteomics

Successful application of cross-linking combined with mass spectrometry for structural proteomics demands specifically designed cross-linking reagents to address challenges in the detection and assignment of cross-links. A combination of affinity enrichment, isotopic coding, and cleavage of the cross-linker is beneficial for detection and identification of the peptide cross-links. Here we describe a novel cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), that allows affinity enrichment of cross-linker-containing peptides with avidin. Affinity enrichment eliminates interfering non-cross-linked peptides and allows the researcher to focus on the analysis of the cross-linked peptides. CBDPS is also isotopically coded and CID-cleavable. The cleaved fragments still contain a portion of the isotopic label and can therefore be distinguished from unlabeled fragments by their distinct isotopic signatures in the MS/MS spectra. This cleavage information has been incorporated into a program for the automatic analysis of the MS/MS spectra of the cross-links. This allows rapid determination of cross-link type in addition to facilitating identification of the individual peptides constituting the interpeptide cross-links. Thus, affinity enrichment combined with isotopic coding and CID cleavage allows in-depth mass spectrometric analysis of the peptide cross-links. We have characterized the performance of CBDPS on the 120-kDa protein heterodimer of HIV reverse transcriptase. Successful application of cross-linking combined with mass spectrometry for structural proteomics demands specifically designed cross-linking reagents to address challenges in the detection and assignment of cross-links. A combination of affinity enrichment, isotopic coding, and cleavage of the cross-linker is beneficial for detection and identification of the peptide cross-links. Here we describe a novel cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), that allows affinity enrichment of cross-linker-containing peptides with avidin. Affinity enrichment eliminates interfering non-cross-linked peptides and allows the researcher to focus on the analysis of the cross-linked peptides. CBDPS is also isotopically coded and CID-cleavable. The cleaved fragments still contain a portion of the isotopic label and can therefore be distinguished from unlabeled fragments by their distinct isotopic signatures in the MS/MS spectra. This cleavage information has been incorporated into a program for the automatic analysis of the MS/MS spectra of the cross-links. This allows rapid determination of cross-link type in addition to facilitating identification of the individual peptides constituting the interpeptide cross-links. Thus, affinity enrichment combined with isotopic coding and CID cleavage allows in-depth mass spectrometric analysis of the peptide cross-links. We have characterized the performance of CBDPS on the 120-kDa protein heterodimer of HIV reverse transcriptase. Cross-linking combined with mass spectrometric analysis is an attractive technique for obtaining structural information on proteins and protein complexes (1.Sinz A. Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions.Mass Spectrom. Rev. 2006; 25: 663-682Crossref PubMed Scopus (525) Google Scholar). Cross-linked proteins can be enzymatically digested, and the cross-linked peptides (cross-links) obtained can be analyzed by mass spectrometry to identify both the cross-linked peptides and the site of cross-linking. Unfortunately, ion signals from the cross-links are usually overwhelmed by ion signals from non-cross-linked or “free” peptides and often difficult to detect and to assign. The most straightforward way of simplifying the mixture is by using cross-linking reagents with affinity tags such as biotin that allow selective enrichment of all of the cross-linker-containing peptides in the digest. Despite the added inconvenience of the synthesis, several biotinylated cross-linking reagents have been reported recently (2.Chu F. Mahrus S. Craik C.S. Burlingame A.L. Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces.J. Am. Chem. Soc. 2006; 128: 10362-10363Crossref PubMed Scopus (50) Google Scholar, 3.Hurst G.B. Lankford T.K. Kennel S.J. Mass spectrometric detection of affinity purified crosslinked peptides.J. Am. Soc. Mass Spectrom. 2004; 15: 832-839Crossref PubMed Scopus (62) Google Scholar, 4.Fujii N. Jacobsen R.B. Wood N.L. Schoeniger J.S. Guy R.K. A novel protein crosslinking reagent for the determination of moderate resolution protein structures by mass spectrometry (MS3-D).Bioorg. Med. Chem. Lett. 2004; 14: 427-429Crossref PubMed Scopus (33) Google Scholar, 5.Petrotchenko E.V. Thomas J.M. Borchers C.H. A collection of novel isotopically-coded crosslinkers for structural proteomics.in: presented at the 56th Annual Conference on Mass Spectrometry and Allied Topics. June 1–5, 2008Google Scholar, 6.Kang S. Mou L. Lanman J. Velu S. Brouillette W.J. Prevelige Jr., P.E. Synthesis of biotin-tagged chemical cross-linkers and their applications for mass spectrometry.Rapid Commun. Mass Spectrom. 2009; 23: 1719-1726Crossref PubMed Scopus (44) Google Scholar). Even after the selective enrichment step, the detection of cross-links is still challenging. The most popular solution for facilitating specific detection of cross-links is isotopic coding of the cross-linking reagents (7.Müller 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: 1927-1934Crossref PubMed Scopus (187) Google Scholar). To enhance the signals from cross-links and to increase the likelihood of their identification, cross-links can be separated from the interfering free (non-cross-linked) peptides, thereby increasing their absolute and relative abundance and simplifying the subsequent mass spectrometric analysis. The final challenge results from the combinatorial nature of the possible interpeptide cross-links that complicates their assignment for complex protein systems even after affinity purification. This can be addressed by using cleavable cross-linkers, allowing sequencing of the individual peptides comprising the interpeptide cross-link. It would therefore be desirable to combine all of these useful features into a single cross-linking reagent that would simultaneously facilitate detection, enrichment, and identification of cross-links. Cross-linking experiments normally produce a considerable number of cross-links (over 100 cross-links for a midsize protein), most of which are the less informative dead-end cross-links (attached through only one reactive group) and intrapeptide cross-links (two cross-linked residues within one peptide) instead of the more informative interpeptide cross-links that provide distance information. Rapid and automatic discrimination of cross-link type is therefore another desirable feature of a combined cross-linking/mass spectrometry analysis. To discriminate dead-end from interpeptide cross-links using cross-linking reagents, several methods have been proposed, including conducting the cross-linking reaction in H218O water (2.Chu F. Mahrus S. Craik C.S. Burlingame A.L. Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces.J. Am. Chem. Soc. 2006; 128: 10362-10363Crossref PubMed Scopus (50) Google Scholar, 8.Seebacher J. Mallick P. Zhang N. Eddes J.S. Aebersold R. Gelb M.H. Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing.J. Proteome Res. 2006; 5: 2270-2282Crossref PubMed Scopus (103) Google Scholar), leading to incorporation of the oxygen atom from water during the hydrolysis of the unreacted active group of the cross-linker, which leads to a characteristic 2-Da shift for dead-end cross-links. This type of isotopic coding has been successfully combined with biotin tagging (2.Chu F. Mahrus S. Craik C.S. Burlingame A.L. Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces.J. Am. Chem. Soc. 2006; 128: 10362-10363Crossref PubMed Scopus (50) Google Scholar, 3.Hurst G.B. Lankford T.K. Kennel S.J. Mass spectrometric detection of affinity purified crosslinked peptides.J. Am. Soc. Mass Spectrom. 2004; 15: 832-839Crossref PubMed Scopus (62) Google Scholar), enabling the researcher to focus the analysis on the affinity-purified cross-links. Isotopic coding of the cross-linking reagents has also been combined with cleavage of the cross-linkers to allow the researcher to distinguish between the three cross-link types and to identify the individual peptides forming the cross-link (9.Petrotchenko E.V. Olkhovik V.K. Borchers C.H. Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes.Mol. Cell. Proteomics. 2005; 4: 1167-1179Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 10.Petrotchenko E.V. Xiao K. Cable J. Chen Y. Dokholyan N.V. Borchers C.H. BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics.Mol. Cell. Proteomics. 2009; 8: 273-286Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Because the cleaved fragments are still isotopically coded, they can be easily detected in the spectra, and their relationships to the uncleaved parent cross-link can be determined based on mass differences. Cleavage of cross-linkers can be either done chemically (10.Petrotchenko E.V. Xiao K. Cable J. Chen Y. Dokholyan N.V. Borchers C.H. BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics.Mol. Cell. Proteomics. 2009; 8: 273-286Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), photoinduced (10.Petrotchenko E.V. Xiao K. Cable J. Chen Y. Dokholyan N.V. Borchers C.H. BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics.Mol. Cell. Proteomics. 2009; 8: 273-286Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), or done using CID (11.Soderblom E.J. Bobay B.G. Cavanagh J. Goshe M.B. Tandem mass spectrometry acquisition approaches to enhance identification of protein-protein interactions using low-energy collision-induced dissociative chemical crosslinking reagents.Rapid Commun. Mass Spectrom. 2007; 21: 3395-3408Crossref PubMed Scopus (47) Google Scholar, 12.Soderblom E.J. Goshe M.B. Collision-induced dissociative chemical cross-linking reagents and methodology: applications to protein structural characterization using tandem mass spectrometry analysis.Anal. Chem. 2006; 78: 8059-8068Crossref PubMed Scopus (91) Google Scholar, 13.Lu Y. Tanasova M. Borhan B. Reid G.E. Ionic reagent for controlling the gas-phase fragmentation reactions of cross-linked peptides.Anal. Chem. 2008; 80: 9279-9287Crossref PubMed Scopus (53) Google Scholar). CID cleavage of cross-links has the advantage that the cleavage reaction occurs inside the mass spectrometer and can be performed individually by automatically mass-selecting each cross-link using an “include list.” The combination of affinity purification with isotopic coding and CID cleavage enables automated comprehensive mass spectrometric analysis of a large number of peptide cross-links. Here we describe a novel cross-linker that combines all three of these features for the rapid automated analysis of the peptide cross-links: isotopic coding, CID cleavage, and biotin affinity tagging of the cross-linker. These three features ensure effective enrichment, confident detection, rapid automated cross-link type determination, and identification of the individual peptides composing each interpeptide cross-link. Here we describe this isotopically coded CID-cleavable biotinylated cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), 1The abbreviations used are:CBDPScyanurbiotindipropionylsuccinimideaaamino acid(s)BS3bis(sulfosuccinimidyl) suberate. 1The abbreviations used are:CBDPScyanurbiotindipropionylsuccinimideaaamino acid(s)BS3bis(sulfosuccinimidyl) suberate. as well as the specialized software (ICCLMSMS) that has been developed for the automated processing of the MS/MS spectra from these cross-links. cyanurbiotindipropionylsuccinimide amino acid(s) bis(sulfosuccinimidyl) suberate. cyanurbiotindipropionylsuccinimide amino acid(s) bis(sulfosuccinimidyl) suberate. All materials were from Sigma-Aldrich unless noted otherwise. 0.1 mmol of cyanuric chloride was incubated with 0.1 mmol of hydrazidobiotin in 50% acetone or 1:1 acetone:D2O for 30 min on ice. To these reaction mixtures, solutions containing 0.2 mmol of mercaptopropionic acid prepared from either bromopropionic acid-H4 or bromopropionic acid-D4 (C/D/N Isotopes) and thiourea (10.Petrotchenko E.V. Xiao K. Cable J. Chen Y. Dokholyan N.V. Borchers C.H. BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics.Mol. Cell. Proteomics. 2009; 8: 273-286Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) were added. Reaction mixtures were neutralized by addition of 10 m NaOH or NaOD, respectively, and incubated at 75 °C for 30 min in open air. Residues were brought to 1 ml with water or D2O, and cyanurbiotindipropionate was precipitated by the addition of hydrochloric acid. The precipitates were washed twice with water or D2O and dried in vacuo. The resulting dipropionic acids were activated in dimethyl sulfoxide with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide. The reaction mixtures were then filtered, and the combined filtrates were partitioned in 1:10 chloroform:water. The chloroform layer was collected and dried in vacuo. The overall yield was 30%. A 1:1 molar ratio mixture of CBDPS-H8/D8 is now available from www.creativemolecules.com. Model cross-linked peptides were prepared by incubating the synthetic peptide Ac-TRTESTDIKRASSREADYLINKER (Creative Molecules Inc.) with an equimolar amount of CBDPS-H8/D8 followed by enzymatic digestion with trypsin. The resulting peptide mixture was separated by reversed-phase HPLC. 1-ml fractions were collected and analyzed by MALDI-MS. Fractions containing dead-end and interpeptide cross-links were used for the affinity enrichment experiments. A 1-μl aliquot of the chromatographic fraction containing the interpeptide Lys-Lys cross-link TESTDIKR-CBDP-EADYLINKER was mixed with 10 μl of a 1 mg/ml tryptic digest of bovine serum albumin (BSA) in phosphate-buffered saline (PBS), pH 7.2. The mixture was affinity-purified with 10 μl of monomeric avidin-agarose bead slurry (Pierce). Beads were washed with PBS and then with water, and the affinity-bound material was eluted with 0.1% TFA and with 0.1% TFA, 50% acetonitrile. Aliquots from the loading, flow-through, wash, and elution fractions were desalted using C18 Zip-Tips (Millipore) and were analyzed by MALDI-MS. A 10-μl aliquot of a 1 mg/ml solution of HIV reverse transcriptase (HIV-RT) (Worthington Biochemical Corp.) in PBS was mixed with 1 μl of a 0.5 mm CBDPS-H8/D8 solution in water prepared from a 50 mm stock solution of the cross-linker. The final concentration of the cross-linker reagent was chosen based on the preliminary titration of the reaction mixture where the intensity of the cross-linked heterodimer band on SDS-PAGE was optimized without the appearance of any from cross-linked The pH of the mixture was to by the addition of 0.2 m The reaction mixture was incubated for 30 min at °C and 50 mm The cross-linked proteins were then with sequencing at °C at a The resulting peptide mixture was affinity-purified using monomeric as for the The elution fraction was by and separated by reversed-phase on a with an C18 and a in with C18 Inc.) 100 This was at a of using a from to The was at a using a The were with 1 mg/ml acid solution in 0.1% TFA, 50% and analyzed by and using a MS/MS spectra were using 50 and a MS/MS where fragmentation was desirable for the cross-link spectra were using and a MS/MS The mass spectra were analyzed using the software E.V. Borchers C.H. isotopically-coded cleavable crosslinking analysis PubMed Scopus Google Scholar) and the program The program with as the for the can be used as an to possible of a cross-link The mass spectra from each chromatographic fraction were for using the program of The mass obtained was used as an for automatic MS/MS The MS/MS spectra were for isotopic signatures characteristic of cross-links using the which were determined to be interpeptide cross-links by the were using the of the software the of the cross-linker, we advantage of the of the cyanuric We were to the of hydrazidobiotin with cyanuric chloride in an equimolar reaction mixture without considerable of the and of the resulting were with mercaptopropionic which was obtained by reaction of or bromopropionic acid with followed by hydrolysis with The obtained dipropionic acid was activated with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide. This in a cross-linking reagent with a of with The of the reagent is determined by the N-hydroxysuccinimide which with the on and the S. R. Chemical cross-linking with a on amino acid Mass Spectrom. 2009; PubMed Scopus Google Scholar, S. A. and of chemical cross-linking with N-hydroxysuccinimide Chem. 2008; PubMed Scopus Google Scholar). The interaction is used for specific enrichment of of from complex a biotin group into the of the cross-linker reagent an affinity that can be used for specific or of all the containing a cross-linker Cross-linking of proteins with a biotinylated cross-linker followed by enzymatic digest leads to of a complex peptide mixture with all of the cross-linker-containing peptide with of biotinylated peptides followed by peptides and the subsequent elution of the biotinylated peptides results in specific purification of the peptide cross-links. The of this for the mass spectrometric analysis are the of ion by the of the more free peptides, and the specific enrichment and concentration of the peptide cross-links. affinity purification of the peptide as that this purification most of the interfering free peptides. This to a for the cross-link in the elution was in the peptide mixture The of the affinity-purified fraction allows subsequent MS/MS analysis of the peptide cross-links. The was in the of the cross-link were detected in the fraction of the cross-linked proteins 30 were detected in the elution fraction from the of the in the single mass which of the digest MS/MS analysis of the interpeptide CBDPS cross-link that cleavage of the cross-linker was the cleavage and MS/MS CID and The cleavage occurs to the cyanuric group to the cleavage of the dead-end cross-links can be distinguished by a of signals in the MS/MS spectra, at a mass the which to a peptide that the of the cross-linker The characteristic to the cleaved portion of the cross-linker. This often an ion that results from the of the ion during cleavage as abundance signals in the MS/MS spectra of dead-end cross-links were an at to the ion with the cross-linker and an at to the cross-linker that was cleaved at the of the and signals from the of the cross-linker, which with the from the of these that the from the of the cross-linker the were in the of the containing the of the cross-linker. of of from the of the cross-link and in that often occurs between the of the cross-linker can only be in the of the cross-linker where leads to an characteristic isotopic which an for the that the and a 1 in the cleaved portion of the cross-link that the the of the CID cleavage of this cross-linker be the of a this an and specific for and cross-link Cleavage of the CBDPS cross-link a isotopic of from which the individual peptides constituting the interpeptide cross-link can easily be Thus, this cross-linker the most of isotopically coded cleavable cross-linkers, allowing the identification of the peptides comprising the interpeptide cross-links. This the cross-link which are the in cross-linking of the combinatorial nature of interpeptide cross-links as We have reported chemically and isotopically coded cross-linking reagents (9.Petrotchenko E.V. Olkhovik V.K. Borchers C.H. Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes.Mol. Cell. Proteomics. 2005; 4: 1167-1179Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 10.Petrotchenko E.V. Xiao K. Cable J. Chen Y. Dokholyan N.V. Borchers C.H. BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics.Mol. Cell. Proteomics. 2009; 8: 273-286Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The advantage of CID-cleavable cross-linkers with of cleavage is that each cross-link can be and from the in the allowing individual cleavage and mass spectrometric analysis of each the of the cleaved peptides that the cross-link with fragments from each individual the peptides can be analyzed by on the of an ion provide information on each individual peptide that the cross-link The mass relationships between the ion were used to a program for the analysis of the MS/MS spectra of isotopically coded CID-cleavable the mass for an of MS/MS spectra can be automatically for the presence of and and from dead-end a of with one and the in mass the ion mass that are characteristic for the dead-end of that to the individual peptides resulting from CID cleavage of the and that to the ion mass and that the individual cleaved peptides constituting the interpeptide cross-link. 1 and are used to distinguish dead-end cross-links from interpeptide and and are used for the interpeptide cross-links. for this combination of a and efficient of these cross-link types in an automated The program can be used for any isotopically coded CID-cleavable cross-linker and is available on the To the performance of CBDPS on the protein of and was cross-linked using this The cross-linking reaction mixture was with and with trypsin. The was to be for the of any biotinylated reagent and any with cross-links during the subsequent affinity enrichment is the amount of to be used for purification relative to the amount of the protein To of all of the biotinylated the of or and the number of residues in the protein can be and equimolar of and residues can be used as a for the affinity purification The resulting peptide mixture was then affinity-purified with monomeric and analyzed by MALDI-MS. in the of the peptide the for affinity purification of the CBDPS cross-links to enrichment of the cross-links in the and of the peptides, thereby resulting in a containing only signals from the of which were in the fraction to the purification (two cross-links were detected without affinity enrichment, were detected after affinity of the affinity-purified cross-links by the detection of the cross-links by The mass spectra of the fractions contain only signals from cross-links Mass spectra of the chromatographic fractions were then for using the program within the software The resulting mass was used as the for automatic acquisition of MS/MS spectra. A of were detected and analyzed by The MS/MS spectra were with the program to and cross-linker fragments signals for dead-end cross-links and to identify the of interpeptide cross-links. The dead-end cross-links were from and the interpeptide cross-link were on the of the cross-link the of the individual peptides from the CID cleavage, and using the program within which was with for the cross-links were as as tryptic three as and as interpeptide cross-links that were CBDPS tryptic interpeptide acid 1 also be or on the of mass between these residues in the are and and mass for and and amino acid residues of the cross-linked peptides, 1 and of cleaved This also be or on the of mass between these residues in the are in a new and and mass for and and amino acid residues of the cross-linked peptides, 1 and of cleaved The cross-links were to the of the Y. J. K. Jr., of reverse transcriptase at A of for and 4: Full Text Full Text PDF PubMed Scopus Google All the between the cross-linked were in with the of the cross-linker and with results E.V. Borchers C.H. of a combination of isotopically coded cross-linkers and isotopically coded reagents for selective identification of Chem. PubMed Scopus Google Scholar), of this new cross-linking of the cross-links were from new cross-links were using this new of the cross-links from either the of one cross-link is only with the and the A where the distance between these residues would be the bis(sulfosuccinimidyl) cross-link between and can only be in the A the cross-link between and can only be in the This of is possible only the of the of the were CBDPS three possible new cross-links to a that be in only one of is within the possible distance by and this cross-link can in either the combination of the specific features of CBDPS this reagent attractive for cross-linking cross-links can be distinguished on the of their isotopic signatures in the spectra and cleaved by CID to produce individual peptides that are from fragments on the of their new isotopic signatures in the MS/MS spectra. We successfully this using analysis of the cross-linked The is in as in with to be to possible in of the and isotopic of the cross-links. This be in a of the cross-linker with isotopic instead of a in the cross-linking allows one to the of the individual peptides composing the interpeptide cross-links in fragmentation of the peptide The relative of individual peptide instead of peptide fragments for cross-link identification is an we have that the of the individual peptides, with mass the combinatorial with interpeptide cross-links and the number of possible for a cross-link. These can be by peptide which can be obtained by CID fragmentation of the cross-links or by possible by analysis of the cleaved peptides. Here we have a novel cross-linker, for structural CBDPS combines three of we are features for mass spectrometric analysis of the peptide cross-links: isotopic coding, and affinity affinity enrichment of cross-linked peptides using each cross-link can be individually cleaved by Cleavage of the cross-linker leads to of easily isotopically coded of the peptides that the cross-link. This allows automated analysis of the MS/MS spectra for which the isotopic coding of the the assignment of the peptides.

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