Peptide backbone fragmentation initiated by side-chain loss at cysteine residue in matrix- assisted laser desorption/ionization in-source decay mass spectrometry Daiki Asakawa, a,b† * Nicolas Smargiasso, a Loïc Quinton a and Edwin De Pauw a Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is initiated by hydrogen transfer from matrix molecules to the carbonyl oxygen of peptide backbone with subsequent radical-induced cleavage leading to c 0 /z• fragments pair. MALDI-ISD is a very powerful method to obtain long sequence tags from proteins or to do de novo sequencing of peptides. Besides classical fragmentation, MALDI-ISD also shows specific fragments for which the mechanism of formation enlightened the MALDI-ISD process. In this study, the MALDI-ISD mechanism is reviewed, and a specific mechanism is studied in details: the N-terminal side of Cys residue (Xxx-Cys) is described to promote the generation of c 0 and w fragments in MALDI-ISD. Our data suggest that for sequences containing Xxx-Cys motifs, the N–C a bond cleavage occurs following the hydrogen attachment to the thiol group of Cys side-chain. The c•/w fragments pair is formed by side-chain loss of the Cys residue with subsequent radical-induced cleavage at the N–C a bond located at the left side (N-terminal direction) of the Cys residue. This fragmentation pathway preferentially occurs at free Cys residue and is suppressed when the cysteines are involved in disulfide bonds. Hydrogen attachment to alkylated Cys residues using iodoacetamide gives free Cys residue by the loss of •CH 2 CONH 2 radical. The presence of alkylated Cys residue also suppress the formation of c•/w fragments pair via the (C b )-centered radical, whereas w fragment is still observed as intense signal. In this case, the z• fragment formed by hydrogen attachment of carbonyl oxygen followed side-chain loss at alkylated Cys leads to a w fragment. Hydrogen attachment on peptide backbone and side-chain of Cys residue occurs therefore competi- tively during MALDI-ISD process. Copyright © 2013 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article. Keywords: top-down sequencing; hydrogen attachment; (C b )-centered radical; 1,5-diaminonapthalene; reduction and alkylation Introduction Mass spectrometry (MS) is now considered as a major analytical tool for proteins and peptides analysis and matrix-assisted laser desorption/ionization (MALDI) [1] is traditionally used for that purpose. One of the main advantages of MALDI is that it yields mainly to singly-charged ions without abundant fragmentation. Peptide mass fingerprinting (PMF) with MALDI-MS is now a common approach for the characterization of isolated proteins by the analysis of their peptide digest. [2,3] Tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) is a commonly available fragmentation method for the characterization of peptides. [4] However, CID is not applicable for a top-down sequencing of proteins, because of the low fragmentation efficiency of large proteins. In addition, the presence of disulfide bridges induces the generation of fewer fragments that are complex to annotate. In contrast to CID, laser- induced fragmentation occurring during the MALDI processes called in-source decay (ISD) has been used for that purpose, even if the fragmentation yield is generally low. MALDI-ISD is a fast fragmentation occurring in the MALDI ion source. It mainly leads to specificN–C a bond cleavage on peptide backbone and was discovered by Brown and Lennon. [5] The fragmentation is less prone to specific cleavages, such as cleavages due to preferential sites of protonation in classical MS/MS by CID. MALDI-ISD has been used to identify the sequence of intact proteins without any enzyme digestion, [6–8] including the localization of posttransla- tional modifications. [9] It is also a useful method for MALDI imaging approach. [10,11] In contrast, metastable decay fragments are not categorized as ISD when the decay rate constant is too low for the reaction to occur in the source. This late fragmentation is called post-source decay (PSD) as it occurs in the mass spectrometer after the ion extraction, between the ion source and the detector. [12] PSD shares some similarities with CID and can be explained by vibrational activation. [13] Although PSD is not applicable directly for a top-down protein sequencing as well as CID, combination of ISD and PSD/CID, i.e. isolation of ISD ions followed by PSD/ * Correspondence to: Daiki Asakawa, Chemistry Department and GIGA-R, Mass Spectrometry Laboratory, University of Liège, Belgium. E-mail: dasakawa@yokohama-cu.ac.jp, dasakawa@mch.pref.osaka.jp † present Address: Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan a Chemistry Department and GIGA-R, Mass Spectrometry Laboratory, University of Liège, Belgium b Graduate School in Nanobioscience, Mass Spectrometry Laboratory, Yokohama City University, Japan J. Mass Spectrom. 2013, 48, 352–360 Copyright © 2013 John Wiley & Sons, Ltd. Special feature: perspective Received: 28 November 2012 Revised: 4 February 2013 Accepted: 5 February 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3182 352