Use of Proteinase K Nonspecific Digestion for Selective and Comprehensive Identification of Interpeptide Cross-links: Application to Prion Proteins
2012; Elsevier BV; Volume: 11; Issue: 7 Linguagem: Inglês
10.1074/mcp.m111.013524
ISSN1535-9484
AutoresEvgeniy V. Petrotchenko, Jason J. Serpa, Darryl B. Hardie, Mark Berjanskii, Bow P. Suriyamongkol, David S. Wishart, Christoph H. Borchers,
Tópico(s)Protein Structure and Dynamics
ResumoChemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrPC) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrPC and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90–124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys185–Lys220 cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers. Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrPC) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrPC and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90–124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys185–Lys220 cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers. Structural proteomics, which combines protein chemistry methods with modern mass spectrometry techniques for protein and peptides, is an emerging technology in structural biology. One of the tools for modern structural proteomics is chemical cross-linking combined with mass spectrometry (1Sinz A. Chemical cross-linking and mass spectrometry for investigation of protein-protein interactions.Mass Spectrom. Protein Interact. 2007; : 83-107Google Scholar, 2Chakravarti B. Lewis S.J. Chakravarti D.N. Raval A. Three dimensional structures of proteins and protein complexes from chemical cross-linking and mass spectrometry: A biochemical and computational overview.Curr. Proteomics. 2006; 3: 1-21Crossref Google Scholar, 3Sinz A. Chemical crosslinking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes.J. Mass Spectrom. 2003; 38: 1225-1237Crossref PubMed Scopus (250) Google Scholar, 4Petrotchenko E.V. Borchers C.H. Crosslinking combined with mass spectrometry for structural proteomics.Mass Spectrom. Rev. 2010; 29: 862-876Crossref PubMed Scopus (154) Google Scholar, 5Young M.M. Tang N. Hempel J.C. Oshiro C.M. Taylor E.W. Kuntz I.D. Gibson B.W. Dollinger G. High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5802-5806Crossref PubMed Scopus (392) Google Scholar). Cross-linking analysis provides the distance between two cross-linked amino acid residues, whereas mass spectrometry provides information on which residues are connected. The basis of this method is a chemical reaction of the cross-linking reagent with functional groups on a protein, leading to the formation of a covalent bond between each end of the reagent and the protein. The distance between two cross-linked sites is determined by the length of the spacer in the cross-linking reagent. Thus, identification of the cross-linked sites on a protein or a protein complex provides spatial information and distance constraints for the two amino acid residues that are connected by the cross-linker. The use of chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics (4Petrotchenko E.V. Borchers C.H. Crosslinking combined with mass spectrometry for structural proteomics.Mass Spectrom. Rev. 2010; 29: 862-876Crossref PubMed Scopus (154) Google Scholar, 6Back J.W. de Jong L. Muijsers A.O. de Koster C.G. Chemical cross-linking and mass spectrometry for protein structural modeling.J. Mol. Biol. 2003; 331: 303-313Crossref PubMed Scopus (202) Google Scholar, 7Sinz 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, 8Lee Y.J. Mass spectrometric analysis of cross-linking sites for the structure of proteins and protein complexes.Mol. BioSystems. 2008; 4: 816-823Crossref PubMed Scopus (68) Google Scholar). In recent years, we and others have been developing an array of cross-linking and modification reagents, software, and methods specifically designed for protein cross-linking experiments combined with MS analysis (9Petrotchenko E.V. Serpa J.J. Borchers C.H. Use of a combination of isotopically coded cross-linkers and isotopically coded N-terminal modification reagents for selective identification of inter-peptide crosslinks.Anal. Chem. 2010; 82: 817-823Crossref PubMed Scopus (28) Google Scholar, 10Petrotchenko E.V. Serpa J.J. Borchers C.H. An isotopically-coded CID-cleavable biotinylated crosslinker for structural proteomics.Mol. Cell. Proteomics. 2011; 10.1074/mcp.M110.001420Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 11Petrotchenko E.V. Borchers C.H. ICC-CLASS: Isotopically-coded cleavable crosslinking analysis suite.BMC Bioinformatics. 2010; 11: 6410.1186/1471-2105-11-64Crossref PubMed Scopus (49) Google Scholar, 12Petrotchenko 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, 13Petrotchenko E.V. Thomas J.M. Borchers C.H. A collection of novel isotopically-coded crosslinkers for structural proteomics.Presented at the 56th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics. Denver, CO, 2008Google Scholar, 14Petrotchenko E.V. Borchers C.H. Cross-linking as a tool to examine protein complexes: Examples of cross-linking strategies and computational modeling.in: Chance M. Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics. Wiley and Sons, Hoboken, NJ2008Google Scholar, 15Petrotchenko E.V. Olkhovik V.K. Borchers C.H. Isotopically-coded cleavable crosslinker 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, 16Leitner A. Walzthoeni T. Kahraman A. Herzog F. Rinner O. Beck M. Aebersold R. Probing native protein structures by chemical cross-linking, mass spectrometry and bioinformatics.Mol. Cell. Proteomics. 2010; 9: 1634-1649Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 17Rappsilber J. Mann M. What does it mean to identify a protein in proteomics?.Trends Biochem. Sci. 2002; 27: 74-78Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 18Singh P. Panchaud A. Goodlett D.R. Chemical cross-linking and mass spectrometry as a low-resolution protein structure determination technique.Anal. Chem. 2010; 82: 2636-2642Crossref PubMed Scopus (90) Google Scholar). For mass spectrometric determination of the cross-linking sites, the cross-linked protein is typically digested with proteolytic enzymes, and the peptides are examined by mass spectrometry. Challenges associated with the cross-linking technique include finding the cross-linked peptides in the complex mixture that often results from this digestion. For this reason, isotopically coded cross-linkers are often used to produce a distinct "signature" for the cross-links in the mass spectra (19Müller 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). A cross-linker that can be cleaved with collision-induced dissociation is also highly beneficial because it facilitates identification of the linked peptides and determination of the cross-linking sites by MS/MS sequencing, after a peak corresponding to a cross-link (a cross-linked pair of peptides) has been found (20Soderblom 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). To simplify the mass spectra and enhance the detectablility of the cross-linked peptides, affinity enrichment can be used (21Hurst 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). Affinity purification is also important for enriching the sample in cross-linker-containing peptides. Cross-linkers with a biotin moiety incorporated in them, for example, can be enriched on avidin beads. Even these methods, however, will enrich the sample in all types of cross-links: dead-end and intrapeptide, as well as the more desirable interpeptide cross-links. Another challenge in cross-linking studies is the lack of suitable cleavage sites. Although trypsin is the most widely used enzyme for proteomics studies, the existence of only a few tryptic cleavage sites in some proteins, including prions, limit the power of this traditional proteolytic enzyme, especially when used with amino-reactive cross-linkers, which target the same amino acids as trypsin and prevent cleavage at the modified sites. This often results in large interpeptide cross-links that are above the optimum range for successful detection by MALDI-MS and MALDI-MS/MS fragmentation. Double digestion with two different high specificity enzymes may somewhat reduce the overall size of the cross-links, but in many cases the interpeptide cross-links still remain fairly large. In this paper, we examine the use of cyanurbiotindipropionylsuccinimide (CBDPS), 1The abbreviations used are:CBDPScyanurbiotindipropionylsuccinimidePrPCnative (noninfectious) prion proteinPrPββ-oligomeric form of the prion proteinEDC1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride]. 1The abbreviations used are:CBDPScyanurbiotindipropionylsuccinimidePrPCnative (noninfectious) prion proteinPrPββ-oligomeric form of the prion proteinEDC1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride]. an isotopically labeled affinity-purifiable CID-cleavable cross-linker, in combination with a nonspecific enzyme, proteinase K, to overcome all of these limitations. Proteinase K, a nonspecific protease, has already been successfully used for the mass spectrometric characterization of difficult-to-digest proteins (22Wu C.C. MacCoss M.J. Howell K.E. Yates 3rd, J.R. A method for the comprehensive proteomic analysis of membrane proteins.Nat. Biotechnol. 2003; 21: 532-538Crossref PubMed Scopus (606) Google Scholar, 23Papasotiriou D.G. Jaskolla T.W. Markoutsa S. Baeumlisberger D. Karas M. Meyer B. Peptide mass fingerprinting after less specific in-gel proteolysis using MALDI-LTQ-Orbitrap and 4-chloro-α-cyanocinnamic acid.J. Proteome Res. 2010; 9: 2619-2629Crossref PubMed Scopus (10) Google Scholar), and for the localization of disulfide bonds (24Hwa J. Klein-Seetharaman J. Khorana H.G. Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4872-4876Crossref PubMed Scopus (64) Google Scholar). Proteinase K digestion of a CBDPS-linked model peptide helped reveal the cleavage characteristics of this enzyme/cross-linker combination. We then applied the CBDPS/proteinase K method to two different conformations of the prion protein: the native noninfectious form (PrPC) and the β-oligomeric form (PrPβ). cyanurbiotindipropionylsuccinimide native (noninfectious) prion protein β-oligomeric form of the prion protein 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride]. cyanurbiotindipropionylsuccinimide native (noninfectious) prion protein β-oligomeric form of the prion protein 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride]. All materials were from Sigma-Aldrich unless otherwise noted. The model peptide Ac-TRTESTDIKRASSREADYLINKER (Creative Molecules Inc., Victoria, Canada) was cross-linked with an equimolar amount of CBDPS-H8/D8 reagent (cyanurbiotindipropionylsuccinimide; Creative Molecules Inc.), as previously described (10Petrotchenko E.V. Serpa J.J. Borchers C.H. An isotopically-coded CID-cleavable biotinylated crosslinker for structural proteomics.Mol. Cell. Proteomics. 2011; 10.1074/mcp.M110.001420Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The pH of the mixture was adjusted to 8.0–8.5 by the addition of 0.2 m Na2HPO4. The reaction mixture was incubated for 30 min at 25 °C and quenched with 50 mm ammonium bicarbonate. The cross-linked peptide was then digested with proteinase K (Invitrogen) for 1 h at 37 °C at a 1:1 (w/w) enzyme:substrate ratio. All of the prion constructs were obtained from Dr. Wishart's laboratory at the University of Alberta via the Prion Protein and Plasmid Production Platform Facility (PrP5) (www.prp5.ca) of PrioNet Canada (http://www.prionetcanada.ca/). Specifically, a synthetic gene corresponding to the Syrian hamster prion protein sequence 90–232 (shPrP90–232) with a 23-residue N-terminal fusion tag containing His6 and a thrombin cleavage site (MGSSHHHHHHSSGLVPRGSHMLE) was synthesized by DNA 2.0. The gene was cloned into a pET15b expression vector between XhoI and EcoRI restriction sites and heat shock transformed into Escherichia coli strain BL21 (DE3). For expression, the transformed cells were grown in 100 ml of LB plus 100 μg/ml ampicillin overnight to generate a starter culture. Between 1.5 and 2% of this starter culture was then used to inoculate 1 liter of LB medium (giving a starting A600 of 0.1). The cells were allowed to reach an A600 between 0.8 and 1.0 before induction with 1 mm isopropyl β-d-thiogalactopyranoside. Twelve to eighteen hours later, the cells were harvested by centrifugation at 5000 rpm for 30 min at 4 °C. The inclusion of the His6 tag afforded a standardized nickel affinity purification strategy previously described by Zahn et al. (27Zahn R. von Schroetter C. Wüthrich K. Human prion proteins expressed in Escherichia coli and purified by high-affinity column refolding.FEBS Lett. 1997; 417: 400-404Crossref PubMed Scopus (248) Google Scholar). PrPβ was generated by exposing the native PrPC protein to low pH (1.0) for 1 h and then dialyzing it back to pH 5.5. The details of the purification and PrPC to PrPβ conversion protocol have been described elsewhere (28Bjorndahl T.C. Zhou G.P. Liu X. Perez-Pineiro R. Semenchenko V. Saleem F. Acharya S. Bujold A. Sobsey C.A. Wishart D.S. Detailed biophysical characterization of the acid-induced PrPc to PrPb conversion process.Biochemistry. 2011; 50: 1162-1173Crossref PubMed Scopus (0) Google Scholar). A 10-μl aliquot of a 1 mg/ml solution of PrPC and PrPβ 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 in DMSO. 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 dimer band of the β-oligomeric form of the prion protein (PrPβ) on SDS-PAGE was maximized, without the appearance of any nonspecific higher oligomeric cross-linked products of the native noninfectious prion protein (PrPC). The pH of the mixture was adjusted to 8.0–8.5 by the addition of 0.2 m Na2HPO4. The reaction mixture was incubated for 30 min at 25 °C and quenched with 50 mm ammonium bicarbonate. The cross-linked proteins were then digested with proteinase K, for 2.5 h at 37 °C at a 1:1 (w/w) enzyme:substrate ratio. A protease inhibitor mixture (P8465; Sigma-Aldrich) was added to the resulting peptide mixture. Monomeric avidin beads (ThermoFisher, Thermo Scientific, Mississauga, Canada) were used for the affinity enrichment. The mixture was affinity-purified with 80 μl of monomeric avidin-agarose beads slurry. The beads were washed three times with 120 μl of 100 mM ammonium acetate and then with water, and the affinity-bound material was eluted with 100 μl of 0.1% TFA and 100 μl of 0.1%TFA, 50% acetonitrile. Aliquots from the loading, flow-through, wash, and elution fractions were desalted using Zip-Tips C18 (Millipore) and were analyzed by MALDI-MS. ESI mass spectrometric analyses were performed on the Orbitrap Velos (Thermo Scientific) mass spectrometer, in the positive ion mode. The PK digest was loaded onto a C18 Zip-tip (Millipore, Billerica, MA) and eluted with 60% methanol, 0.1% formic acid. Nano-ESI of the eluate was performed using Proxeon nanoES capillaries (Thermo Scientific) at 1.6-kV spray voltage. ESI-MS spectra were acquired manually over an m/z range of 400–2000, using FT detection profile mode with a resolution of 60,000 and an isolation width of 1 Da. ESI-MS/MS spectra were manually acquired using CID fragmentation in the FT profile mode, with an isolation width of 2 Da, a collision energy setting of 30%, and a resolution of 60,000. Thermo Scientific Excalibur software, version 2.1.0 QF03489 build 1140, was used for the data acquisition. Thermo Proteome Discoverer 1.3, version 1.3.0.339 was used to generate the .mgf files. The eluted fractions were combined, concentrated by lyophilization, and separated by nano-flow reversed phase HPLC on an Ultimate nano-LC system (Dionex/LC Packings, Sunnyvale, CA) equipped with an LC Packings (Sunnyvale, CA) 0.3 × 5 mm C18 PepMap trapping column (5-μm particle size, 100 Å pore size), and a 75-μm × 15-cm capillary column packed in-house with Magic C18 Aq (Michrom Bioresources Inc., Auburn, CA) particles (5 μm, 100 Å). This capillary LC system was operated at a flow rate of 300 nl/min, using a 55-min gradient from 5 to 60% acetonitrile (0.1% TFA). The column effluent was spotted at 1-min intervals (300 nl/spot) onto a stainless steel MALDI target using a Shimadzu (Kyoto, Japan) spotter. The spots were dried, overlaid with 4 mg/ml α-cyano-4-hydroxycinnamic acid matrix solution in 0.1% TFA, 50% acetonitrile, and analyzed by MALDI-MS and MS/MS, using a 4800 MALDI-TOF/TOF (AB Sciex, Concord, Canada). MS/MS spectra were acquired using "CID off," 50-FHWM gate width, and a 1-kV MSMS method. In cases where additional fragmentation was desirable for the unambiguous cross-link assignments, spectra were reacquired using "CID on" and a 2-kV MSMS method. The mass spectra were analyzed using the ICC-CLASS software (11Petrotchenko E.V. Borchers C.H. ICC-CLASS: Isotopically-coded cleavable crosslinking analysis suite.BMC Bioinformatics. 2010; 11: 6410.1186/1471-2105-11-64Crossref PubMed Scopus (49) Google Scholar) and the ICCLMSMS program (9Petrotchenko E.V. Serpa J.J. Borchers C.H. Use of a combination of isotopically coded cross-linkers and isotopically coded N-terminal modification reagents for selective identification of inter-peptide crosslinks.Anal. Chem. 2010; 82: 817-823Crossref PubMed Scopus (28) Google Scholar) (version date: August 1, 2011; available free at www.creativemolecules.com/CM_Software.htm). Mass spectra peaklists were generated using Data Explorer Version 4.0 (AB Sciex). The mass spectra from each chromatographic fraction were searched for the undeuterated/deuterated CBDPS-cross-linked (D0/D8) doublets using the DX program of ICC-CLASS, with peak intensity cutoff of 50 and a mass tolerance setting of 0.01 Da for the 8.05824 Da theoretical mass difference between the masses of the light and heavy isotopes. The D0/D8 mass list obtained was used as inclusion list for automatic MS/MS spectra acquisition. The acquired MS/MS spectra were searched for isotopic signatures characteristic of CID cleavage of cross-links 401 Da apart, using the ICCLMSMS program. Interpeptide cross-links were recognized based on their CID cleavage patterns and were identified by the DXMSMS module of the ICC-CLASS software package based on the cross-link mass, the masses of the CID cleavage products, and the masses of the fragments of the cross-link, using the following settings: 100-ppm mass tolerance for the precursor mass, 300-ppm mass tolerance for MS/MS fragment ions, all possible cleavage sites, and KYST as allowed cross-linking sites. The NMR structure of hamster prion protein (Protein Data Bank code 1B10) (29James T.L. Liu H. Ulyanov N.B. Farr-Jones S. Zhang H. Donne D.G. Kaneko K. Groth D. Mehlhorn I. Prusiner S.B. Cohen F.E. PDB ID 1B10: Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10086-10091Crossref PubMed Scopus (429) Google Scholar) was used as the initial template, which covers only residues 125–231. The missing residues from the flexible N terminus (Gly68–Gly124) were modeled in an extended conformation and were connected to the initial template via superpositioning, followed by energy minimization of these residues using XPLOR-NIH (30Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. The Xplor-NIH NMR molecular structure determination package.J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1865) Google Scholar). MS cross-linking data was converted into distance constraints between the side chain nitrogen atoms of lysine residues, with an upper limit of 14 Å. For Gly68, the N-terminal residue of the N-terminal fusion tag, the position of the N-terminal nitrogen was used for the distance calculations. An ensemble of 200 models was generated by molecular dynamics in Cartesian space using XPLOR-NIH (30Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. The Xplor-NIH NMR molecular structure determination package.J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1865) Google Scholar). Specifically, the positions of the N-terminal region (Gly68–Gly124) with respect to the initial template (Leu125–Gly228) was optimized with a simulated annealing protocol that included 6000 steps at 1000 K and 3000 cooling steps from 1000 K to 0 K, followed by 1000 steps of Powell minimization (31Powell M.J. On search directions for minimization algorithms.Math. Programming. 1973; 4: 193-201Crossref Scopus (214) Google Scholar). In this initial model, the positions of the backbone atoms of the initial template were kept fixed. The structures were visualized using the molecular modeling program MOLMOL (32Koradi R. Billeter M. Wüthrich K. MOLMOL: A program for display and analysis of macromolecular structures.J. Mol. Graphics Modelling. 1996; 14 (29-32): 51-55Crossref Scopus (6489) Google Scholar). To characterize the types of peptides produced by proteinase K digestion of the interpeptide cross-links, we analyzed a digest of the model peptide, Ac-TRTESTDIKRASSREADYLINKER, cross-linked with the CBDPS reagent (Fig. 1). CBDPS is homo-bifunctional NHS-based amine-reactive cross-linker, possessing additional features useful for mass spectomeric analyses, such as isotopic coding, CID cleavage, and affinity purification (10Petrotchenko E.V. Serpa J.J. Borchers C.H. An isotopically-coded CID-cleavable biotinylated crosslinker for structural proteomics.Mol. Cell. Proteomics. 2011; 10.1074/mcp.M110.001420Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Previous studies have shown that intrapeptide cross-links are generated by cross-linking this peptide with CBDPS. Digestion of this cross-linked peptide with proteinase K, a nonspecific enzyme, resulted in sets of short, related, interpeptide CBDPS cross-links, all of which contain the same cross-linked pair of lysine residues. As can be seen from Fig. 1A, proteinase K cleaves the peptides at or near the cross-linked residue, leaving two to four residues of each peptide still attached to the cross-linker. This usually results in a set of related isotopically labeled cross-links for each cross-linked pair of amino acids. In the example shown in Fig. 1A, there are multiple cross-links containing the same pair of cross-linked residues; for example, the cross-linked Lys-Lys pair (Lys9–Lys22) is found six times in the spectrum. Multiple cross-links often differed by one amino acid residue, creating a "sequence ladder" around the cross-linking site, which can be used as an additional verification tool for the cross-link assignment.Fig. 1MALDI-MS and MS/MS analysis of the test peptide (Ac-TRTESDKIRASSREADYLINKER) cross-linked with CBDPS-H8/D8, followed by proteinase K digestion. A, MALDI-MS spectrum of the proteinase K digest. Inset, cross-links appear in the spectrum as doublets of signals 8.05 Da apart (theoretical), because of the isotopic coding of the cross-linking reagent. B, MS/MS spectrum of the m/z 1200 interpeptide IK-KER cross-link. (Note: masses of the isotopically coded peptide and fragment ion pairs are denoted as vertically aligned pairs of numbers corresponding to light and heavy isotopic forms; fragment ions are labeled with subscripts α or β, denoting the peptide from which they originated; fragments containing portions of the cross-linker and counterpart peptide are labeled with the subscript cl.) C, ESI-MS spectrum of proteinase K digest. Inset, expanded region of the doubly charged molecular ion of the cross-link, showing the isotopic labeling. D, ESI-MS/MS spectrum of the proteinase K digest. In positive ion ESI MS/MS, the fragmentation depends on the charge state of the precursor ion. The ESI-MS/MS spectrum of the +2 charge state of the precursor ion is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because of the molecular weight of the CBDPS cross-linker (∼500 Da), the interpeptide cross-links fall within the optimum mass range (1100–1500 Da), for MALDI-MS detection and MS/MS sequencing. An example of an MS/MS spectrum is shown in Fig. 1B. Use of the CID-cleavable cross-linker CBDPS allowed determination of the two component peptides forming the cross-link by MS/MS. The relatively short sequences from each cross-linked peptide simplify the MS/MS spectra and facilitate the sequence assignment of the cross-links. It should be noted that the proteinase K cleavage does not proceed completely to give only crosslinked dipeptides or K-CBDP-K crosslinks, possibly due to the experimental conditions, or steric hindrance from the crosslinker or from the connected peptide. Interestingly, in a paper on membrane proteins (done at higher pH) this "laddering" was also observed (22Wu C.C. MacCoss M.J. Howell K.E. Yates 3rd, J.R. A method for the comprehensive proteomic analysis of membrane
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