Exopeptidase digestion in combination with field desorption mass spectrometry for amino acid sequence determination

Abstract

Numerous studies have been devoted in the last years to the development of mass spectrometric methods for the sequence determination of peptides [ 1,2]. Most advanced among this work has been so far the rigorous chemical derivatization of oligopeptides to achieve sufficient volatility for the application of conventional (electron impact, EI) mass spectrometry [2-41. For example, the analysis by gas chromatography-mass spectrometry (GC-MS) of mixtures of oligopeptide fragments derivatized after chemical or enzymatic hydrolysis of polypeptides has been successfully used for sequence determinations [2,5]. Major limitations of this approach are that only small peptide derivatives are amenable to GC-MS, and the relatively large amount of original peptide needed. More recently, mass spectra of underivatized peptides have been obtained by field desorption (FD) mass spectrometry [6], a technique that does not require any volatility of the sample to be ionized. It was shown that FD mass spectra of oligopeptides usually exhibit molecular ions (e.g., protonated, MH’ ions) of high intensity, with relatively little fragmentation [7,8]. While studies of thermally or collisioninduced fragmentation in FD spectra of peptides [9-l l] so far indicate only limited utility for obtaining structural information, the possibility of analysing peptide mixtures by means of the predominant molecular ions has been suggested [ 121. FD mass spectrometry in combination with Edman degradation-subtractive analysis has been reported [ 131. the available enzymes (e.g., contamination with endopeptidase or different types of exopeptidase activity), and the problem of determining repetitive amino acid residues have limited their application so far. Attempts to overcome these difficulties have been made such as the addition of endopeptidase inhibitors [ 14,151 and solid-phase sequencing with exopeptidases [ 161. However, the characteristic feature of the inhomogeneous kinetics of exopeptidase digestion which, in principle, should match well the capability of FD mass spectrometry to analyse peptide mixtures prompted our interest to test the combination of both methods for peptide sequencing. Here, fast and unequivocal sequence determinations were obtained for 2 oligopeptides by direct FD mass spectral analysis of aliquots taken from partial aminoand carboxypeptidase digests The finding that FD mass spectra of exopeptidase digests provided reliable sequence data without any separation step required, and with amounts of material at the sub-nanomole level indicate that this combination may prove to be a valuable approach among the instrumentarium for the sequence determination of peptides.