The exploitation of selective cleavage of singly protonated peptide ions adjacent to aspartic acid residues using a quadrupole orthogonal time-of-flight mass spectrometer equipped with a matrix-assisted laser desorption/ionization source Anthony. G. Sullivan a , Francesco L. Brancia a , Richard Tyldesley b , Robert Bateman b , Khushwant Sidhu c , Simon J. Hubbard c , Stephen G. Oliver d , Simon. J. Gaskell a, * a Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Manchester M60 1QD, United Kingdom b Micromass UK Ltd., Manchester M23 9LE, United Kingdom c Department of Biomolecular Sciences, UMIST, Manchester M60 1QD, United Kingdom d School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Received 17 January 2001; accepted 13 February 2001 Abstract A series of singly charged tryptic peptide ions were analyzed by tandem mass spectrometry using a quadrupole/time-of- flight instrument equipped with a matrix-assisted laser desorption/ionization source. Highly selective cleavage C-terminal to aspartate, but not glutamate, residues was observed for C-terminal arginine-containing peptides, consistent with earlier findings. Increasing the basicity of C-terminal lysine residues by conversion to homoarginine promoted selective cleavage adjacent to aspartate residues. In contrast, reducing the basicity of C-terminal arginine residues by conversion to dimethylpyrimidylornithine abolished selective backbone cleavage and allowed the formation of multiple sequence ions. The increase in database search selectivity incorporating the single-residue sequence tag information revealed by aspartate-specific cleavage was determined by simulations using the yeast proteome, it was shown that an average of 83% of proteins can be identified on the basis of the mass of a single tryptic peptide together with knowledge of the presence and location of an aspartate residue. (Int J Mass Spectrom 210/211 (2001) 665– 676) © 2001 Elsevier Science B.V. Keywords: Orthogonal-time-of-flight; Peptide fragmentation; Database searching; Proteomics 1. Introduction The combination of matrix-assisted laser desorp- tion/ionization (MALDI) with hybrid tandem instru- ments of quadrupole/time-of-flight design provides new opportunities for the study of the collisionally activated decomposition (CAD) of ions produced by MALDI [1,2]. The MALDI technique is now very widely used in the context of proteomics, that is, the analysis of multiple constituents of the full protein complement a cell type or organism. Typi- cally, protein isolation is followed by enzymatic (usually tryptic) digestion followed by mass profil- ing of the digest and automated comparison with pre- * Corresponding author. E-mail: Simon.Gaskell@umist.ac.uk Dedicated to Professor Nico Nibbering on the occasion of his retirement. 1387-3806/01/$20.00 © 2001 Elsevier Science B.V. All rights reserved PII S1387-3806(01)00430-4 International Journal of Mass Spectrometry 210/211 (2001) 665– 676 www.elsevier.com/locate/ijms