JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2006; 41: 939–949 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jms.1053 C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus Zhong Li, Talat Yalcin and Carolyn J. Cassady * Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487 Received 27 February 2006; Accepted 9 May 2006 Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H] - and [M - 2H] 2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18 O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode. Copyright  2006 John Wiley & Sons, Ltd. KEYWORDS: C-terminal residue loss; deprotonated peptides; SORI-CID; PSD; acidic residues INTRODUCTION Mass spectrometry is an essential tool for gaining peptide sequence information. 1–3 Most efforts have involved pro- tonated peptides or metallated peptides with the charge on the peptide ion being positive. In these studies, selec- tive cleavages caused by the presence of specific amino acid residues are an interesting aspect of dissociation. One example is the truncation of peptide ions by elimination of the C-terminal residue. This process was first observed for alkali metal-cationized peptides. 4–7 The resulting product ion is a shortened peptide, [b n1 C 17 C alkali] C , where n represents the number of amino acid residues in the parent peptide. Glish and coworkers 5,8 found that these truncated peptide ions can further lose their own C-terminal residues if sufficient internal energy is applied in MS n experiments. This repeated loss of C-terminal residues can facilitate the assignment of the peptide sequence. L Correspondence to: Carolyn J. Cassady, Department of Chemistry, Box 870336, The University of Alabama, Tuscaloosa, AL 35487. E-mail: cassady@bama.ua.edu Several mechanisms have been proposed to rationalize the process of C-terminal residue loss from alkali metal- cationized peptides. 4,9–14 Adams and coworkers 11,12 sug- gested that the amide carbon is nucleophilically attacked by the C-terminal carboxylic acid group after the alkali metal cation coordinates with the carbonyl oxygen of the amide. 12,15 The resulting cleavages would produce a truncated pep- tide ion and two neutral products. Gross and coworkers 9,13 suggested that alkali metal ions may also coordinate to a C-terminal carboxylate to form a zwitterion structure. This is supported by the fact that peptides containing ester or amide groups at the C-terminus 9,11 do not fragment to produce [b n1 C 17 C alkali] C ; that is, this fragmentation requires a carboxylic acid group, –COOH, at the C-terminus. In addi- tion, Lebrilla and coworkers 4 proposed that the mechanism may involve a rearrangement to an anhydride intermediate that fragments to eliminate the C-terminal residue. C-Terminal residue loss is less common for protonated peptides 7,16 – 19 than for alkali metal-cationized peptides. For protonated peptides, the process is most often found when the sequence contains basic residues (e.g. arginine) 7,16,17 or has a site of fixed positive charge (e.g. phosphonium salts). 6,19 Copyright  2006 John Wiley & Sons, Ltd.