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• W2418700897 abstract "The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation. The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation. The complement system performs immune surveillance, enabling rapid recognition and clearance of invading pathogens as well as apoptotic cells and particles threatening homeostasis (1.Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat. Immunol. 2010; 11: 785-797Crossref PubMed Scopus (2460) Google Scholar). Multiple complement-activation pathways converge at the assembly of C3 convertases (2.Walport M.J. Complement. First of two parts.N. Engl. J. Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2388) Google Scholar). These bimolecular proteolytic enzymes excise the anaphylatoxin domain (ANA 1The abbreviations used are:ANAanaphylatoxinBS3Bis[sulfosuccinimidyl] suberateEMElectron microscopyFDRFalse discovery rateHCDHigher energy collision induced dissociationLC-MS/MSLiquid chromatography tandem mass spectrometryLTQLinear trap quadrupoleMS2Tandem mass spectrometryPSMpeptide-spectrum-matchQCLMSQuantitative cross-linking/mass spectrometrySCXStrong cation exchange., corresponding to C3a) from the complement component C3 (184 kDa) leaving its activated form, C3b (175 kDa) (Fig. 1A). C3b can covalently attach, via a nascently exposed and activated thioester, to any nearby surface (3.Law S.K. Lichtenberg N.A. Levine R.P. Evidence for an ester linkage between the labile binding site of C3b and receptive surfaces.J. Immunol. 1979; 123: 1388-1394PubMed Google Scholar, 4.Tack B.F. Harrison R.A. Janatova J. Thomas M.L. Prahl J.W. Evidence for presence of an internal thiolester bond in third component of human complement.Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 5764-5768Crossref PubMed Scopus (262) Google Scholar) whereupon it undergoes rapid amplification (2.Walport M.J. Complement. First of two parts.N. Engl. J. Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2388) Google Scholar). anaphylatoxin Bis[sulfosuccinimidyl] suberate Electron microscopy False discovery rate Higher energy collision induced dissociation Liquid chromatography tandem mass spectrometry Linear trap quadrupole Tandem mass spectrometry peptide-spectrum-match Quantitative cross-linking/mass spectrometry Strong cation exchange. The complement system responds very swiftly to pathogens, independently of antibodies, due primarily to its “alternative pathway” of activation. This is initiated by spontaneous, although rare, conformational changes within C3 that are concerted with hydrolysis of its constitutively buried thioester linkage (5.Isaac L. Aivazian D. Taniguchi-Sidle A. Ebanks R.O. Farah C.S. Florido M.P. Pangburn M.K. Isenman D.E. Native conformations of human complement components C3 and C4 show different dependencies on thioester formation.Biochem. J. 1998; 329: 705-712Crossref PubMed Scopus (13) Google Scholar). Identical conformational changes accompany attack of the thioester by amines (6.Pangburn M.K. Muller-Eberhard H.J. Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement.J. Exp. Med. 1980; 152: 1102-1114Crossref PubMed Scopus (228) Google Scholar). The continuously and ubiquitously generated stable product, C3(H2O) (iC3 or C3N) does not bind to surfaces (as it no longer possesses a thioester group). Interestingly, C3(H2O) has been inferred to resemble C3b in many of its functional and structural features, despite its retention of the ANA (7.Gros P. Milder F.J. Janssen B.J. Complement driven by conformational changes.Nat. Rev. Immunol. 2008; 8: 48-58Crossref PubMed Scopus (234) Google Scholar, 8.Isenman D.E. Kells D.I. Cooper N.R. Muller-Eberhard H.J. Pangburn M.K. Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties.Biochemistry. 1981; 20: 4458-4467Crossref PubMed Scopus (118) Google Scholar, 9.Li K. Gor J. Perkins S.J. Self-association and domain rearrangements between complement C3 and C3u provide insight into the activation mechanism of C3.Biochem. J. 2010; 431: 63-72Crossref PubMed Scopus (24) Google Scholar, 10.Pangburn M.K. Schreiber R.D. Muller-Eberhard H.J. Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.J. Exp. Med. 1981; 154: 856-867Crossref PubMed Scopus (321) Google Scholar) (Fig. 1A, 1B). The mature C3 molecule consists of two polypeptide chains (residues 1–645 in the β-chain and residues 650–1641 in the α-chain). A metaphor of a puppeteer holding a puppet has been used to describe the crystal structure of C3 (11.Janssen B.J. Christodoulidou A. McCarthy A. Lambris J.D. Gros P. Structure of C3b reveals conformational changes that underlie complement activity.Nature. 2006; 444: 213-216Crossref PubMed Scopus (294) Google Scholar) (supplemental Fig. S1 in Supplemental File). Macroglobulin domains (MGs) 1–6 and a “linking region” (LNK) adopt a key-ring like arrangement that forms the body of the puppeteer whereas MG7, MG8, and ANA form its shoulders, and a C345C domain equates to its head, joined to MG8 by an “anchor” region (the neck). A thioester-containing domain (TED) is the puppet, held at shoulder height by a CUB domain that forms the arm of the puppeteer. MGs 1–5, LNK, and half of MG6 are contributed by the β-chain whereas the remaining domains are coming from the α-chain. Comparing the crystal structures of C3 and C3b revealed significant domain rearrangements between them (11.Janssen B.J. Christodoulidou A. McCarthy A. Lambris J.D. Gros P. Structure of C3b reveals conformational changes that underlie complement activity.Nature. 2006; 444: 213-216Crossref PubMed Scopus (294) Google Scholar). Most dramatically, the CUB arm swings away from the shoulders toward the “feet” of the puppeteer (supplemental Fig. S1). As a result, the TED (i.e. the puppet) rotates and is repositioned. This is accompanied by exposure and activation of the thioester group, allowing attachment of C3b to surface-borne nucleophiles. The crystal structure of C3(H2O) has not been reported. New binding sites for complement components and cell-surface receptors are created in both nascent C3b and C3(H2O) (7.Gros P. Milder F.J. Janssen B.J. Complement driven by conformational changes.Nat. Rev. Immunol. 2008; 8: 48-58Crossref PubMed Scopus (234) Google Scholar, 12.Alsenz J. Becherer J.D. Nilsson B. Lambris J.D. Structural and functional analysis of C3 using monoclonal antibodies.Curr. Top. Microbiol. Immunol. 1990; 153: 235-248PubMed Google Scholar, 13.Becherer J.D. Alsenz J. Lambris J.D. Molecular aspects of C3 interactions and structural/functional analysis of C3 from different species.Curr. Top. Microbiol. Immunol. 1990; 153: 45-72PubMed Google Scholar, 14.Becherer J.D. Lambris J.D. Identification of the C3b receptor-binding domain in third component of complement.J. Biol. Chem. 1988; 263: 14586-14591Abstract Full Text PDF PubMed Google Scholar, 15.Forneris F. Ricklin D. Wu J. Tzekou A. Wallace R.S. Lambris J.D. Gros P. Structures of C3b in complex with factors B and D give insight into complement convertase formation.Science. 2010; 330: 1816-1820Crossref PubMed Scopus (194) Google Scholar, 16.Lambris J.D. Avila D. Becherer J.D. Muller-Eberhard H.J. A discontinuous factor H binding site in the third component of complement as delineated by synthetic peptides.J. Biol. Chem. 1988; 263: 12147-12150Abstract Full Text PDF PubMed Google Scholar, 17.Lambris J.D. Lao Z. Oglesby T.J. Atkinson J.P. Hack C.E. Becherer J.D. Dissection of CR1, factor H, membrane cofactor protein, and factor B binding and functional sites in the third complement component.J. Immunol. 1996; 156: 4821-4832PubMed Google Scholar, 18.Morgan H.P. Schmidt C.Q. Guariento M. Blaum B.S. Gillespie D. Herbert A.P. Kavanagh D. Mertens H.D. Svergun D.I. Johansson C.M. Uhrin D. Barlow P.N. Hannan J.P. Structural basis for engagement by complement factor H of C3b on a self surface.Nat. Struct. Mol. Biol. 2011; 18: 463-470Crossref PubMed Scopus (191) Google Scholar). Both proteins bind factor B that is subsequently cleaved to Bb. Importantly, both the resultant C3bBb and C3(H2O)Bb complexes are C3 convertases, generating further molecules of C3b and thereby stoking a positive-feedback loop. Because C3(H2O) (unlike C3b) is a spontaneously arising product of C3 domain rearrangements and thioester hydrolysis, C3(H2O)Bb (rather than C3bBb) is the initiating convertase of the alternative pathway of complement activation. Thus the constitutive presence of C3(H2O) ensures the alternative pathway can be activated quickly and indiscriminately allowing a rapid response to any cell not protected by the appropriate regulatory molecules such as factor H. Inappropriate regulation of complement activity is linked to many autoimmune, inflammatory and ischemia/reperfusion (I/R) injury-related diseases (19.Ricklin D. Lambris J.D. Complement-targeted therapeutics.Nat. Biotechnol. 2007; 25: 1265-1275Crossref PubMed Scopus (389) Google Scholar). It has been shown that hydrolysis of the thioester in C3 alone does necessarily result in transition to active C3(H2O) (20.Pangburn M.K. Spontaneous reformation of the intramolecular thioester in complement protein C3 and low temperature capture of a conformational intermediate capable of reformation.J. Biol. Chem. 1992; 267: 8584-8590Abstract Full Text PDF PubMed Google Scholar). Despite use of diverse methodologies (7.Gros P. Milder F.J. Janssen B.J. Complement driven by conformational changes.Nat. Rev. Immunol. 2008; 8: 48-58Crossref PubMed Scopus (234) Google Scholar, 9.Li K. Gor J. Perkins S.J. Self-association and domain rearrangements between complement C3 and C3u provide insight into the activation mechanism of C3.Biochem. J. 2010; 431: 63-72Crossref PubMed Scopus (24) Google Scholar, 10.Pangburn M.K. Schreiber R.D. Muller-Eberhard H.J. Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.J. Exp. Med. 1981; 154: 856-867Crossref PubMed Scopus (321) Google Scholar, 11.Janssen B.J. Christodoulidou A. McCarthy A. Lambris J.D. Gros P. Structure of C3b reveals conformational changes that underlie complement activity.Nature. 2006; 444: 213-216Crossref PubMed Scopus (294) Google Scholar, 12.Alsenz J. Becherer J.D. Nilsson B. Lambris J.D. Structural and functional analysis of C3 using monoclonal antibodies.Curr. Top. Microbiol. Immunol. 1990; 153: 235-248PubMed Google Scholar, 13.Becherer J.D. Alsenz J. Lambris J.D. Molecular aspects of C3 interactions and structural/functional analysis of C3 from different species.Curr. Top. Microbiol. Immunol. 1990; 153: 45-72PubMed Google Scholar, 21.Hack C.E. Paardekooper J. Smeenk R.J. Abbink J. Eerenberg A.J. Nuijens J.H. Disruption of the internal thioester bond in the third component of complement (C3) results in the exposure of neodeterminants also present on activation products of C3. An analysis with monoclonal antibodies.J. Immunol. 1988; 141: 1602-1609PubMed Google Scholar, 22.Isenman D.E. Conformational changes accompanying proteolytic cleavage of human complement protein C3b by the regulatory enzyme factor I and its cofactor H. Spectroscopic and enzymological studies.J. Biol. Chem. 1983; 258: 4238-4244Abstract Full Text PDF PubMed Google Scholar, 23.Isenman D.E. Cooper N.R. The structure and function of the third component of human complement–I. The nature and extent of conformational changes accompanying C3 activation.Mol. Immunol. 1981; 18: 331-339Crossref PubMed Scopus (39) Google Scholar, 24.Janssen B.J. Huizinga E.G. Raaijmakers H.C. Roos A. Daha M.R. Nilsson-Ekdahl K. Nilsson B. Gros P. Structures of complement component C3 provide insights into the function and evolution of immunity.Nature. 2005; 437: 505-511Crossref PubMed Scopus (423) Google Scholar, 25.Nishida N. Walz T. Springer T.A. Structural transitions of complement component C3 and its activation products.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19737-19742Crossref PubMed Scopus (102) Google Scholar, 26.Perkins S.J. Sim R.B. Molecular modelling of human complement component C3 and its fragments by solution scattering.Eur. J. Biochem. 1986; 157: 155-168Crossref PubMed Scopus (46) Google Scholar, 27.Winters M.S. Spellman D.S. Lambris J.D. Solvent accessibility of native and hydrolyzed human complement protein 3 analyzed by hydrogen/deuterium exchange and mass spectrometry.J. Immunol. 2005; 174: 3469-3474Crossref PubMed Scopus (22) Google Scholar), the remodeling of domains that underlies spontaneous formation of C3(H2O), and therefore triggers complement, are poorly understood. Current structural models of C3(H2O) rely on epitope-mapping (21.Hack C.E. Paardekooper J. Smeenk R.J. Abbink J. Eerenberg A.J. Nuijens J.H. Disruption of the internal thioester bond in the third component of complement (C3) results in the exposure of neodeterminants also present on activation products of C3. An analysis with monoclonal antibodies.J. Immunol. 1988; 141: 1602-1609PubMed Google Scholar), hydrogen-deuterium exchange (27.Winters M.S. Spellman D.S. Lambris J.D. Solvent accessibility of native and hydrolyzed human complement protein 3 analyzed by hydrogen/deuterium exchange and mass spectrometry.J. Immunol. 2005; 174: 3469-3474Crossref PubMed Scopus (22) Google Scholar), other biophysical solution studies (9.Li K. Gor J. Perkins S.J. Self-association and domain rearrangements between complement C3 and C3u provide insight into the activation mechanism of C3.Biochem. J. 2010; 431: 63-72Crossref PubMed Scopus (24) Google Scholar) and negative-staining EM images (25.Nishida N. Walz T. Springer T.A. Structural transitions of complement component C3 and its activation products.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19737-19742Crossref PubMed Scopus (102) Google Scholar). These indicate a “C3b-like” structure but do not provide direct evidence regarding placements of the ANA and TED relative to specific domains within the shoulders and body of the C3(H2O) molecule. It has been proposed that the ANA domain acts as a safety catch in native C3. Removal of the ANA triggers the dramatic structural transition into C3b (24.Janssen B.J. Huizinga E.G. Raaijmakers H.C. Roos A. Daha M.R. Nilsson-Ekdahl K. Nilsson B. Gros P. Structures of complement component C3 provide insights into the function and evolution of immunity.Nature. 2005; 437: 505-511Crossref PubMed Scopus (423) Google Scholar). More knowledge of the C3(H2O) structure is required to test if the safety catch role of ANA (presumably displaced in C3(H2O) rather than removed, as in C3b) and subsequent domain reconfigurations are general mechanisms, relevant both to the spontaneous but rare hydrolytic C3 to C3(H2O) transition, and to the proteolytic cleavage-dependent but rapid C3 to C3b transition. Further understanding of this event depends on the ability to elucidate, in solution, the dynamic processes whereby the domains of a protein molecule are reorganized, following a triggering event, to form a new stable arrangement. Quantitative cross-linking/mass spectrometry (QCLMS) using isotope-labeled cross-linkers (Fig. 2A) has emerged as a new approach with which to elucidate the details of protein conformational changes (28.Fischer L. Chen Z.A. Rappsilber J. Quantitative cross-linking/mass spectrometry using isotope-labelled cross-linkers.J. Proteomics. 2013; 88: 120-128Crossref PubMed Scopus (108) Google Scholar, 29.Schmidt C. Zhou M. Marriott H. Morgner N. Politis A. Robinson C.V. Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation.Nat. Commun. 2013; 4: 1985Crossref PubMed Scopus (111) Google Scholar, 30.Kukacka Z. Rosulek M. Strohalm M. Kavan D. Novak P. Mapping protein structural changes by quantitative cross-linking.Methods. 2015; 89: 112-120Crossref PubMed Scopus (24) Google Scholar, 31.Walzthoeni T. Joachimiak L.A. Rosenberger G. Rost H.L. Malmstrom L. Leitner A. Frydman J. Aebersold R. xTract: software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry.Nat. Methods. 2015; 12: 1185-1190Crossref PubMed Scopus (65) Google Scholar). In this approach, chemical cross-linking captures proximities between amino acid residues and the residues involved are identified by mass spectrometry. Quantitative comparison of the cross-linking results obtained for two different conformations of a protein allows the details of the conformational change to be elucidated. We have developed a workflow for QCLMS analysis (32.Chen Z.A. Fischer L. Tahir S. Bukowski-Wills J.-C. Barlow P.N. Rappsilber J. Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes.bioRxiv. 2016; (10.1101/055418)Google Scholar). In our benchmark study, we used QCLMS to accurately reveal differences and similarities between C3 and C3b in terms of the spatial arrangements of their domains (32.Chen Z.A. Fischer L. Tahir S. Bukowski-Wills J.-C. Barlow P.N. Rappsilber J. Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes.bioRxiv. 2016; (10.1101/055418)Google Scholar). In another application, this technique successfully revealed conformational changes involved in maturation of the proteasome lid complex (33.Tomko Jr., R.J. Taylor D.W. Chen Z.A. Wang H.W. Rappsilber J. Hochstrasser M. A single alpha helix drives extensive remodeling of the proteasome lid and completion of regulatory particle assembly.Cell. 2015; 163: 432-444Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Here we apply our QCLMS workflow, and an integrative modeling approach, to interrogate the unknown arrangement of domains in, C3(H2O), a key component of the complement alternative pathway. We combined knowledge of the crystal structures of C3 and C3b with QCLMS data sets for C3(H2O), C3 and C3b. We thus generated structural models for the conformational transition of C3 to C3(H2O) that are consistent with other biophysical studies and with previously observed functional similarities and differences between these proteins. Plasma-derived human C3 and C3b were purchased from Complement Technology, Inc., Tyler, TX (and stored at −80 °C). Native C3 was depleted of low amounts of contaminating C3(H2O) using cation-exchange chromatography (34.Sanchez-Corral P. Anton L.C. Alcolea J.M. Marques G. Sanchez A. Vivanco F. Separation of active and inactive forms of the third component of human complement, C3, by fast protein liquid chromatography (FPLC).J. Immunol. Methods. 1989; 122: 105-113Crossref PubMed Scopus (19) Google Scholar). C3(H2O) (i.e. C3(N) in this case but identical to C3(H2O)) was prepared by incubating C3 at 37 °C with 200 mm methylamine (CH3NH2) at pH 8.3 for three hours. The C3(H2O) was then isolated from any other intermediates using cation-exchange chromatography (34.Sanchez-Corral P. Anton L.C. Alcolea J.M. Marques G. Sanchez A. Vivanco F. Separation of active and inactive forms of the third component of human complement, C3, by fast protein liquid chromatography (FPLC).J. Immunol. Methods. 1989; 122: 105-113Crossref PubMed Scopus (19) Google Scholar). Chromatography in both cases was performed using a Mini S PC 3.2/3 column (GE Healthcare, Little Chalfont, UK) at a flow-rate of 500 μl/min at 4 °C and a gradient from 0 to 325 mm NaCl. Immediately after purification, C3, C3(H2O) and C3b samples were exchanged, using 30-kDa molecular weight cutoff (MWCO) filters (Millipore, Cork, Ireland), into cross-linking buffer (20 mm HEPES-KOH, pH 7.8, 20 mm NaCl and 5 mm MgCl2) with a final concentration of 2 μm. C3, C3b and C3(H2O) samples were prepared in two separated batched and used for “experiment I” and “experiment II” respectively. Fifty μg C3, C3b and C3(H2O) were each cross-linked in a volume of 100 μl with either bis[sulfosuccinimidyl] suberate (BS3) (Thermo Fisher Scientific, Rockford, IL) or its deuterated analogue bis[sulfosuccinimidyl] 2,2,7,7-suberate-d4 (BS3-d4) (Thermo Fisher Scientific), at 1:3 protein to cross-linker mass ratio, giving rise to six different protein-cross-linker combinations: C3+BS3, C3+BS3-d4, C3(H2O)+BS3, C3(H2O)+BS3-d4, C3b+BS3 and C3b+BS3-d4. After incubation (two hours) on ice, reactions were quenched with 10 μl 2.5 m ammonium bicarbonate for 45 min on ice. For the monitoring of cross-linking, aliquots containing 5 pmol of cross-linked protein from each of the above six reactions were subjected to SDS-PAGE using a NuPAGE 4–12% Bis-Tris gel (Life Technologies, Carlsbad, CA) and MOPS running buffer (Life Technologies). The protein bands were visualized using the Colloidal Blue Staining Kit (Life Technologies) (Fig. 2B). Cross-linking reactions were repeated for “experiment II” as described for “experiment I”. Each of C3, C3b and C3(H2O) consists of two polypeptide chains linked by a disulfide bond. The monomeric (two-polypeptide chains) product of cross-linked C3, C3b or C3(H2O) was isolated using SDS-PAGE (50 μg was loaded for each). Proteins were in-gel reduced and alkylated, then digested using trypsin following a standard protocol (35.Maiolica A. Cittaro D. Borsotti D. Sennels L. Ciferri C. Tarricone C. Musacchio A. Rappsilber J. Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching.Mol. Cell Proteomics. 2007; 6: 2200-2211Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). For quantitation, equimolar quantities of the tryptic products from the six cross-linked protein samples were mixed pair-wise in four combinations to allow the comparisons of C3(H2O) with C3b and with C3: C3+BS3 and C3(H2O)+BS3-d4 (sample I-1); C3+BS3-d4 and C3(H2O)+BS3 (sample I-2); C3b+BS3 and C3(H2O)+BS3-d4 (sample II-1); and finally C3b+BS3-d4 and C3(H2O)+BS3 (sample II-2) (Fig. 2C). For each of the four samples, a 20 μg (40 μl) aliquot was fractionated using SCX-Stage-Tips (36.Ishihama Y. Rappsilber J. Mann M. Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics.J. Proteome Res. 2006; 5: 988-994Crossref PubMed Scopus (224) Google Scholar) with a small variation of the protocol previously described for linear peptides (37.Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2570) Google Scholar). In short, peptide mixtures were first loaded on a SCX-Stage-Tip in loading buffer (0.5% v/v acetic acid, 20% v/v acetonitrile, 50 mm ammonium acetate). The retained peptides were eluted in two steps, with buffers containing 100 mm ammonium acetate and 500 mm ammonium acetate, into two fractions. These peptide fractions were desalted using C18-StageTips (38.Rappsilber J. Ishihama Y. Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.Anal. Chem. 2003; 75: 663-670Crossref PubMed Scopus (1796) Google Scholar) prior to mass spectrometric analysis. Preparation of four quantitation samples were repeated as described for “experiment I.” A 4-μg (8 μl) aliquot of each sample was desalted using C18-Stage-Tips for mass spectrometric analysis without pre-fractionation. SCX-Stage-Tip fractions were analyzed using a hybrid linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap Velos, Thermo Fisher Scientific, Bremen Germany) applying a “high-high” acquisition strategy. Peptides were separated on an analytical column that was packed with C18 material (ReproSil-Pur C18-AQ 3 μm; Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) in a spray emitter (75-μm inner diameter, 8-μm opening, 250-mm length; New Objectives, Woburn, MA) (39.Ishihama Y. Rappsilber J. Andersen J.S. Mann M. Microcolumns with self-assembled particle frits for proteomics.J. Chromatogr. A. 2002; 979: 233-239Crossref PubMed Scopus (260) Google Scholar). Mobile phase A consisted of water and 0.5% v/v acetic acid. Mobile phase B consisted of acetonitrile and 0.5% v/v acetic acid. Peptides were loaded at a flow-rate of 0.6 μl/min and eluted at 0.3 μl/min using a linear gradient going from 3% mobile phase B to 35% mobile phase B over 130 min, followed by a linear increase from 35% to 80% mobile phase B in 5 mins. The eluted peptides were directly introduced into the mass spectrometer. MS data were acquired in the data-dependent mode. For each acquisition cycle, the mass spectrum was recorded in the Orbitrap with a resolution of 100,000. The eight most intense ions with a precursor charge state 3+ or greater were fragmented in the linear ion trap by collision-induced disassociation (CID). The fragmentation spectra were then recorded in the Orbitrap at a resolution of 7,500. Dynamic exclusion was enabled with single repeat count and 60-s exclusion duration. Non-fractionated peptide samples were analyzed using a hybrid quadrupole-Orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific). Peptides were separated on a reversed-phase analytical column of the same type as described above. Mobile phase A consisted of water and 0.1% v/v formic acid. Mobile phase B consisted of 80% v/v acetonitrile and 0.1% v/v formic acid. Peptides were loaded at a flow rate of 0.5 μl/min and eluted at 0.2 μl/min. The separation gradient consisted of a linear increase from 2% mobile phase B to 40% mobile phase B in 169 min and a subsequent linear increase to 95% B over 11 min. Eluted peptides were directly sprayed into the Q Exactive mass spectrometer. MS data were acquired in the data-dependent mode. For each acquisition cycle, the MS spectrum was recorded in the Orbitrap at 70,000 resolution. The ten most intense ions in the MS spectrum, with a precursor charge state of 3+ or greater, were fragmented by Higher Energy Collision Induced Dissociation (HCD). The fragmentation spectra were thus recorded in the Orbitrap at 35,000 resolution. Dynamic exclusion was enabled, with single-repeat count and a 60-s exclusion duration. The raw mass spectrometric data files were processed into peak lists using MaxQuant version 1.2.2.5 (40.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar) with default parameters, except that “Top MS/MS Peaks per 100 Da” was set to 20. The peak lists were searched against C3 and decoy C3 sequences using Xi software (ERI, Edinburgh) for identification of cross-linked peptides. Search parameters were as follows: MS accuracy, 6 ppm; MS2 accuracy, 20 ppm; enzyme, trypsin; specificity, fully tryptic; allowed number of missed cleavages, four; cross-linker, BS3/BS3-d4; fixed modifications, carbamidomethylation on cysteine; variable modifications, oxidation on methionine; modifications by BS3/BS3-d4 that are hydrolyzed or amidated on the other end. The linkage specificity for BS3 was assumed to be for lysine, serine, threonine, tyrosine and protein N termini. Identified candidates for" @default.
• W2418700897 created "2016-06-24" @default.
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• W2418700897 date "2016-08-01" @default.
• W2418700897 modified "2023-10-18" @default.
• W2418700897 title "Structure of Complement C3(H2O) Revealed By Quantitative Cross-Linking/Mass Spectrometry And Modeling" @default.
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• W2418700897 cites W1530933431 @default.
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• W2418700897 cites W1582008322 @default.
• W2418700897 cites W1582584861 @default.
• W2418700897 cites W1839275362 @default.
• W2418700897 cites W1840984138 @default.
• W2418700897 cites W1963618204 @default.
• W2418700897 cites W1970718615 @default.
• W2418700897 cites W1978496023 @default.
• W2418700897 cites W1985138628 @default.
• W2418700897 cites W1986656413 @default.
• W2418700897 cites W1990643927 @default.
• W2418700897 cites W1998909987 @default.
• W2418700897 cites W2003236418 @default.
• W2418700897 cites W2003427751 @default.
• W2418700897 cites W2007188470 @default.
• W2418700897 cites W2007583388 @default.
• W2418700897 cites W2014850360 @default.
• W2418700897 cites W2023010129 @default.
• W2418700897 cites W2024508275 @default.
• W2418700897 cites W2035450517 @default.
• W2418700897 cites W2036080352 @default.
• W2418700897 cites W2042118984 @default.
• W2418700897 cites W2042777883 @default.
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• W2418700897 cites W2051091550 @default.
• W2418700897 cites W2056236390 @default.
• W2418700897 cites W2068521408 @default.
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• W2418700897 cites W2129754968 @default.
• W2418700897 cites W2131150601 @default.
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• W2418700897 cites W2136648677 @default.
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• W2418700897 cites W2232234141 @default.
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• W2418700897 doi "https://doi.org/10.1074/mcp.m115.056473" @default.
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