Abstract
We report on the characterization by mass spectrometry (MS) of a rapid, reagentless and site-specific cleavage at the N-terminus of the amino acid cysteine (C) in peptides and proteins induced by the thermal decomposition at 220-250 °C for 10 s in solid samples. This thermally induced cleavage at C occurs under the same conditions and simultaneously to our previously reported thermally induced site-specific cleavage at the C-terminus of aspartic acid (D) (Zhang, S.; Basile, F. J. Proteome Res. 2007, 6, (5), 1700-1704). The C cleavage proceeds through cleavage of the nitrogen and α-carbon bond (N-terminus) of cysteine and produces modifications at the cleavage site with an amidation (-1 Da) of the N-terminal thermal decomposition product and a -32 Da mass change of the C-terminal thermal decomposition product, the latter yielding either an alanine or β-alanine residue at the N-terminus site. These modifications were confirmed by off-line thermal decomposition electrospray ionization (ESI)-MS, tandem MS (MS/MS) analyses and accurate mass measurements of standard peptides. Molecular oxygen was found to be required for the thermal decomposition and cleavage at C as it induced an initial cysteine thiol side chain oxidation to sulfinic acid. Similar to the thermally induced D cleavage, missed cleavages at C were also observed. The combined thermally induced digestion process at D and C, termed thermal decomposition/digestion (TDD), was observed on several model proteins tested under ambient conditions and the site-specificity of the method confirmed by MS/MS.