title: Discovery of undefined protein cross-linking chemistry: a comprehensive methodology utilizing 18O-labeling and mass spectrometry.
abstract: Characterization of protein cross-linking, particularly without prior knowledge of the chemical nature and site of cross-linking, poses a significant challenge, because of their intrinsic structural complexity and the lack of a comprehensive analytical approach. Toward this end, we have developed a generally applicable workflow-XChem-Finder-that involves four stages: (1) detection of cross-linked peptides via (18)O-labeling at C-termini; (2) determination of the putative partial sequences of each cross-linked peptide pair using a fragment ion mass database search against known protein sequences coupled with a de novo sequence tag search; (3) extension to full sequences based on protease specificity, the unique combination of mass, and other constraints; and (4) deduction of cross-linking chemistry and site. The mass difference between the sum of two putative full-length peptides and the cross-linked peptide provides the formulas (elemental composition analysis) for the functional groups involved in each cross-linking. Combined with sequence restraint from MS/MS data, plausible cross-linking chemistry and site were inferred, and ultimately confirmed, by matching with all data. Applying our approach to a stressed IgG2 antibody, 10 cross-linked peptides were discovered and found to be connected via thioethers originating from disulfides at locations that had not been previously recognized. Furthermore, once the cross-link chemistry was revealed, a targeted cross-link search yielded 4 additional cross-linked peptides that all contain the C-terminus of the light chain.
authors: Analytical Research and Development, Amgen, One Amgen Center Drive, Thousand Oaks, California 91320, USA.
authors: Min
authors: M
authors: Liu
authors: Zhongqi
authors: Z
authors: Zhang
authors: Tianzhu
authors: T
authors: Zang
authors: Chris
authors: C
authors: Spahr
authors: Janet
authors: J
authors: Cheetham
authors: Da
authors: D
authors: Ren
authors: Zhaohui Sunny
authors: ZS
authors: Zhou
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes; D010446:Peptide Fragments / Q000032:analysis* / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D010103:Oxygen Isotopes; D010446:Peptide Fragments
references: J Biol Chem. 2004 Mar 19;279(12):10901-9
references: 14623886
references: Anal Chem. 2010 Sep 1;82(17):7485-91
references: 20712325
references: Anal Chem. 2005 Oct 1;77(19):6364-73
references: 16194101
references: Anal Chem. 2010 Apr 1;82(7):2636-42
references: 20210330
references: Rapid Commun Mass Spectrom. 2001;15(24):2456-65
references: 11746917
references: Am J Physiol Heart Circ Physiol. 2010 Jul;299(1):H1-9
references: 20472764
references: J Biol Chem. 2008 Jun 6;283(23):16206-15
references: 18339626
references: Bioorg Med Chem Lett. 2000 Nov 6;10(21):2471-5
references: 11078203
references: Anal Chem. 2011 Nov 15;83(22):8642-51
references: 21995278
references: J Mass Spectrom. 2010 Oct;45(10):1212-9
references: 20872902
references: Proteomics. 2008 Feb;8(4):673-85
references: 18214847
references: Anal Chem. 2010 Feb 1;82(3):1090-9
references: 20039682
references: J Biol Chem. 2008 Jun 6;283(23):16194-205
references: 18339624
references: Bioorg Med Chem Lett. 2009 Nov 1;19(21):6200-4
references: 19775890
references: Mol Cell Proteomics. 2010 Aug;9(8):1634-49
references: 20360032
references: Anal Chem. 2008 May 15;80(10):3882-9
references: 18419136
references: Anal Chem. 2013 Feb 19;85(4):2478-85
references: 23316706
references: J Pharm Sci. 2008 Jul;97(7):2426-47
references: 17828757
references: J Proteome Res. 2009 May;8(5):2140-3
references: 19338309
references: Rapid Commun Mass Spectrom. 2004;18(3):319-24
references: 14755618
references: Bioorg Med Chem Lett. 2001 Sep 3;11(17):2331-5
references: 11527725
references: J Pharm Sci. 2009 Sep;98(9):3013-30
references: 18680168
references: J Proteome Res. 2010 Dec 3;9(12):6323-33
references: 20886857
references: Electrophoresis. 1996 May;17(5):945-53
references: 8783021
references: J Proteome Res. 2007 Oct;6(10):3908-17
references: 17854217
references: Anal Chem. 2006 Apr 1;78(7):2145-9
references: 16579592
references: Mass Spectrom Rev. 2007 Jan-Feb;26(1):108-20
references: 17019703
references: Brief Funct Genomic Proteomic. 2009 Mar;8(2):136-44
references: 19151093
references: Eur J Mass Spectrom (Chichester). 2008;14(5):275-80
references: 19023145
references: J Am Soc Mass Spectrom. 2012 Apr;23(4):764-72
references: 22298290
references: Anal Chem. 2009 Jul 1;81(13):5524-32
references: 19496583
references: Anal Chem. 2001 Jul 1;73(13):2836-42
references: 11467524
references: J Am Chem Soc. 2007 Jun 6;129(22):6976-7
references: 17500521
references: Biopolymers. 2007;88(1):55-63
references: 17091497
references: Anal Chem. 2011 Jun 15;83(12):4775-81
references: 21528900
references: Biochem J. 1980 Sep 1;189(3):507-20
references: 7213343
references: Nat Methods. 2008 Apr;5(4):315-8
references: 18327264
references: ChemMedChem. 2007 Aug;2(8):1181-9
references: 17576647
references: J Agric Food Chem. 1999 Apr;47(4):1295-319
references: 10563973
references: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5802-6
references: 10811876
references: Pharm Res. 1992 Nov;9(11):1386-93
references: 1475223
references: FASEB J. 2004 Jul;18(10):1135-7
references: 15132984
references: Rapid Commun Mass Spectrom. 2010 Feb;24(3):267-75
references: 20049891
references: Brain Pathol. 2009 Oct;19(4):612-22
references: 18673368
references: J Agric Food Chem. 1977 Mar-Apr;25(2):245-51
references: 838959
references: Anal Chem. 2005 May 1;77(9):2675-82
references: 15859580
references: Anal Chem. 2008 Nov 15;80(22):8799-806
references: 18947195
references: J Pharm Sci. 2009 Dec;98(12):4511-24
references: 19408342
references: Anal Chem. 2004 Jul 15;76(14):3908-22
references: 15253624
references: J Proteome Res. 2003 Mar-Apr;2(2):147-52
references: 12716128
references: Biopolymers. 2003;71(5):534-51
references: 14635094
references: Proteomics. 2010 Feb;10(4):634-49
references: 19953542
references: Anal Biochem. 2009 Sep 1;392(1):12-21
references: 19457431
references: J Proteome Res. 2011 Mar 4;10(3):923-31
references: 21175198
references: Anal Chem. 2009 Oct 15;81(20):8354-64
references: 19764700
references: J Proteome Res. 2006 Sep;5(9):2270-82
references: 16944939
references: Anal Chem. 2002 Sep 1;74(17):4417-22
references: 12236350
references: Methods Enzymol. 1972;25:424-38
references: 23014423
references: Rapid Commun Mass Spectrom. 2004;18(8):869-76
references: 15095355
references: Anal Chem. 2012 Jan 17;84(2):1056-62
references: 22132761
references: J Proteome Res. 2010 Jul 2;9(7):3583-9
references: 20476776