Imidate-Based Cross-Linkers for Structural Proteomics: Increased Charge of Protein and Peptide Ions and CID and ECD Fragmentation Studies

Autor(es)

Koolen, Hector H. F. ; Gomes, Alexandre F. ; Schwab, Nicolas V. ; Eberlin, Marcos N. ; Gozzo, Fabio C. ^[5]

• Tipo de documento: Artigo Científico
• Fonte: JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY; v. 25, n. 7, p. 1181-1191, JUL 2014.
• Citações Web of Science: 0

Resumo

Chemical cross-linking is an attractive low-resolution technique for structural studies of protein complexes. Distance constraints obtained from cross-linked peptides identified by mass spectrometry (MS) are used to construct and validate protein models. Amidinating cross-linkers such as diethyl suberthioimidate (DEST) have been used successfully in chemical cross-linking experiments. In this work, the application of a commercial diimidate cross-linking reagent, dimethyl suberimidate (DMS), was evaluated with model peptides and proteins. The peptides were designed with acetylated N-termini followed by random sequences containing two Lys residues separated by an Arg residue. After cross-linking reactions, intra- and intermolecular cross-linked species were submitted to CID and ECD dissociations to study their fragmentation features in the gas phase. Fragmentation of intramolecular peptides by collision induced dissociation (CID) demonstrates a unique two-step fragmentation pathway involving formation of a ketimine as intermediate. Electron capture and electron transfer dissociation (ECD and ETD) experiments demonstrated that the cyclic moiety is not dissociated. Intermolecular species demonstrated previously described fragmentation behavior in both CID and ECD experiments. The charge state distributions (CSD) obtained after reaction with DMS were compared with those obtained with disuccinimidyl suberate (DSS). CSDs for peptides and proteins were increased after their reaction with DMS, owing to the higher basicity of DMS modified species. These features were also observed in LC-MS experiments with bovine carbonic anhydrase II (BCA) after cross-linking with DMS and tryptic proteolysis. Cross-linked peptides derived from this protein were identified at high confidence and those species were in agreement with the crystal structure of BCA. (AU)

• Processo FAPESP: 08/57805-2 - Instituto de Bioanalítica
• Beneficiário: Lauro Tatsuo Kubota
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 04/14846-0 - Rede de proteoma do estado de São Paulo
• Beneficiário: Fabio Cesar Gozzo
• Modalidade de apoio: Auxílio à Pesquisa - Programa GENOMA