Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de Novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

A simple mass spectrometric method to sequence a recombinant phosphoenolpyr uvate carboxykinase of known structure and a novel variant of unknown struc ture isolated from Anaerobiospirillum, succiniciproducens and Actinobacillu s succinogenes 130Z, respectively, was evaluated. The proteolytic digests o f the proteins were each chemically derivatized at the N-terminus by additi on of a tris(trimethoxyphenyl)phosphoniumncetyl (TMPP+-Ac) group to produce peptides with a fixed positive charge. The derivatized digests were then p artially separated by reversed-phase high-performance liquid chromatography , The fractions collected were subjected to matrix-assisted laser desorptio n/ionization post-source decay (MALDI/PSD) mass spectrometric analysis. The resulting spectra are sufficiently simple to allow the sequence to be read directly without extensive interpretation. This is in contrast to spectra of underivatized peptides obtained by MALDI/PSD or conventional tandem mass spectrometry, where full sequence interpretation can be challenging, Aided with a set of very simple established rules, it was shown that the sequenc e of TMPP+-Ac derivatives can be derived strictly from predictable fragment ion series, In most cases, this is sufficient to determine extensive, unam biguous, peptide sequences de novo. The partial sequence (35%) of the unkno wn phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes 130Z was obtained entirely by the mass spectrometric method evaluated here, whic h provided the basis for evaluating homology and for the design of oligonuc leotide probes for cloning the corresponding gene. Copyright (C) 1999 John Wiley & Sons, Ltd.