title: Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS.
abstract: We have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (-)-ESI-MS, CID-MS/MS, and MS3experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO-C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids. Graphical Abstract ᅟ.
authors.affiliation: Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120, Halle, Saale, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Hage
authors.affiliation: Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120, Halle, Saale, Germany.
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ihling
authors.affiliation: Institute of Biochemistry, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120, Halle, Saale, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Götze
authors.affiliation: Department of Chemistry, University Cologne, Greinstr. 4, D-50939, Köln, Germany. mathias.schaefer@uni-koeln.de.
authors.forename: Mathias
authors.initials: M
authors.lastname: Schäfer
authors.affiliation: Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120, Halle, Saale, Germany. andrea.sinz@pharmazie.uni-halle.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004952:Esters / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry*; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D003497:Cyclic N-Oxides; D004952:Esters; D005609:Free Radicals; D010455:Peptides; C003959:TEMPO
keywords: Chemical cross-linking; FRIPS; Negative ESI; TEMPO radical; Tandem mass spectrometry