De novo sequencing of disulphide rich peptides using tandem mass spectrometry

Abstract

Peptide based drugs have been shown to have better target selectivity and thereby better safety profile than many small molecule drug candidates. This led many researchers and drug companies to show an increasing interest toward bioactive peptides. Developing an effective analytical method for peptide /protein structural identification is a cornerstone to meet this expanding area of research in this field. De novo sequencing is a method of peptide structural identification without using peptide databases. Compared to the other de novo sequencing methods, mass spectrometric peptide sequence analysis is superior in terms of sensitivity and speed. Peptide structural information is contained in the spectrum produced by tandem mass spectrometers MS/MS. The quality of the data contained in MS/MS spectra depends on the amino acid content of the peptide, the instrumentation and methods used. One commonly used peptide fragmentation method is collision induced dissociation (CID) where the peptide collides with an inert gas. Depending on the amount of energy applied during fragmentation, peptides yield a wide array of product ions. Peptides of known amino acid sequence were used to determine the relationship between precursor ion mass-to- charge-ratio (m/z) and collision energy needed for optimal peptide fragmentation. The energy seems to increase with increasing m/z of the precursor ion. A comparison of CID fragmentation methods in a quadrupole-time-of-flight (QTof) instrument revealed the merit of using a collision energy ramp compared to single collision energy. Evaluation of de novo sequencing accuracy of PEAKS software showed that the program works well for smaller peptides but the accuracy deteriorates with increasing peptide length. The amino acid sequence of an unknown peptide was determined using the PEAKS. The software has identified 84 % the amino acid sequences correctly. The rest 16 % of the residues were confirmed by Edman degradation. The peptide contained three disulphide bridges and identifying cysteine interconnectivity of the peptide is a gap to be addressed in the future. Key words: Collision energy, ramp, PEAKS, de novo sequencing.