A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry

Abstract

A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides.Knowledge of protein sequences is fundamentally important for understanding many physiological processes at the molecular level (1). Tandem mass spectrometry has become an increasingly essential tool for protein and peptide sequencing because of its speed, sensitivity, and applicability to complex mixtures (2). Recently, postsource decay (PSD) matrix-assisted laser desorption ionization (MALDI) mass spectrometry was developed for high-sensitivity peptide sequencing applications (3-8). Subpicomole limits of analysis were reported as a result of the high yield of fragment ions and the high ion transmission inherent with time-of-flight mass spectrometry (5). Kaufmann et al. (5) also noted several problems associated with PSD MALDI sequencing of peptides, including the complexity of the resulting fragmentation patterns and the lack of computer algorithms capable of interpreting the complex spectra. The recent incorporation of delayed extraction (DE) (9, 10), a technique designed to improve precursor-ion mass resolution and mass-measurement accuracy, reduced the rate of PSD fragmentation by at least an order of magnitude. As a result, many low-intensity precursor ions obtained by DE MALDI do not produce enough PSD fragmentation to allow derivation of even short sequence tags (7).Positively charged derivatives have been used in desorption mass spectrometry for many years to improve sensitivity by enhancing ionization efficiencies (11, 12...

Expanding the organismal scope of proteomics: Cross‐species protein identification by mass spectrometry and its implications

“…Upon their collisional fragmentation, tryptic peptides break at their amide bonds between consecutive amino acid residues producing a continuous y-series of fragments from which the amino acid sequence can be read. One method to facilitate this interpretation is to enrich a series of fragments by attaching a strongly positively or strongly negatively charged group to the N-terminus of peptides [16,17]. Another method is to introduce an isotopic label to the C-terminus of peptides by digesting proteins in a buffer containing H 2 18 O (protocols reviewed in [6]) or by CD 3 OH [18].…”

Investigation of the influence of charge derivatization on the fragmentation of multiply protonated peptides

“…The derivatization of the N-terminus with chlorosulfonylacetyl chloride (CSAC) was performed as described previously [25]. A peptide (1 nmol) was dissolved in 2 l 0.02 M sulfonic acid (2 l CSAC in 500 l water).…”

“…This fact confirms the role of the sulfonic acid derivative as it attracts the mobile proton. The dissociation close to the N-terminus has been enhanced, although the influence of the charge state prevents the exclusive formation of y-fragments as obtained from singly charged peptides [22].…”

“…Oxidation of free cysteine to cysteic acid introduced a negative charge on the peptide and changed the dissociation behavior dramatically. As a consequence, a completely different approach has been carried out by Roth et al who derivatized peptides with a negative charge [10,22] to improve the fragmentation of singly charged peptides in MALDI-PSD spectra. The introduction of a negative charge (e.g., sulfonic acid) is counterbalanced by a second mobile proton.…”

Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

“…The main series that could be obtained from peptide fragmentation are summarized in Scheme 1 (Cristoni et al, 2002a). It must also be emphasized that, in this particular case, fragmentation of the underivatized peptide is observed; however, with other peptides very poor fragmentation and little structural information are obtained with the PSD approach without derivatization (Keough, Youngquist, & Lacey, 1999). Figure 4B,C show the effect of two derivatization approaches.…”