Derivatisation of peptides for collision-induced dissociation mass spectrometry

Abstract

This study is concerned with the mass spectrometric analysis of peptides C-terminally derivatised with aminosulphonic acids. The aim was to increase the sensitivity of detection and to simplify the fragmentation patterns obtained upon collision-induced dissociation. The derivatives were analysed using a hybrid magnetic sector/orthogonal time-of-flight mass spectrometer.

A procedure for the C-terminal derivatisation of peptides with 4-aminonaphthalene sulphonic acid was developed. Amino groups were first acetylated to prevent intra molecular reactions. The derivatisation increased the sensitivity of detection by fast-atom bombardment ionisation by one to two orders of magnitude. Collision-induced dissociation at 8 keV collision energy gave product ions formed by charge-remote fragmentations, giving solely C-terminal fragment ions. The absence of product ions formed by other fragmentation processes simplified the spectra and made them less complex than those of the corresponding underivatised peptides. The absence of N-terminal fragment ions made deduction of the amino acid sequence relatively straight-forward.

Conventional electrospray with focal-plane detection permitted detection of the peptide derivatives on a subfemtomol level. Low-energy cone-voltage/collision-induced dissociation and high-energy collision-induced dissociation at a collision energy of 4 keV gave informative spectra. The latter conditions gave fragmentation patterns similar to those obtained with fast-atom bombardment/collision-induced dissociation.

Electrospray/collision-induced dissociation was also performed at 400 eV collision energy using the fourth field-free region collision cell. The fragmentation patterns were highly dependent on the mass of the collision gas. Collisions with helium mainly fragmented the peptide bonds while collisions with xenon fragmented all types of bonds in the peptide backbone and gave side-chain cleavages. For larger peptides (more than 10 amino acid residues) fragment ions arising from cleavage of the peptide bonds dominated. When analysed by nano-electrospray the sensitivity of detection was two orders of magnitude better than with conventional electrospray. Spectra were obtained for derivatised peptides ranging in size from 3 - 15 residues and rules for their fragmentation were established. Small peptide derivatives (3 - 6 residues) could be sequenced by nano electrospray/collision-induced dissociation of a few hundred frnol of sample. Derivatised peptides of unknown sequence obtained by proteolysis of hamster liver aldehyde dehydrogenase were analysed by nano-electrospray/collision-induced dissociation using the fourth field-free region collision cell. Complete or partial amino acid sequence information could be deduced from the spectra. It was concluded that derivatisation with 4-aminonaphthalenesulphonic acid is a useful tool in the de novo sequencing of peptides.