‘De novo’ sequencing by mass spectrometry and confirmation by chemical synthesis
peer-reviewed · Proceedings of the 10th ISMAS Symposium · 2006
| Date | 2006-09-01 |
| Type | peer-reviewed |
| Venue | Proceedings of the 10th ISMAS Symposium |
| Publisher | Indian Society for Mass Spectrometry |
| Contribution | downstream-application |
| Link | https://www.researchgate.net/publication/200723922 |
Abstract
‘De novo’ peptide sequencing of natural peptides using mass spectrometry is becoming increasingly accessible. The peptide sequence is deduced from the mass spectrum through an iterative process by recognizing fragment ions corresponding to the b and y-series. The mass difference between the adjacent ions in each series gives the cleavage mass of the amino acid residue that is lost, leading to the sequence of peptide. Ambiguity in sequencing arise from the presence of isobaric residue masses (leucine, isoleucine, hydroxyproline, lysine and glutamine) and also from presence of residue pairs which differ in mass by 1Da sometimes leading to sequencing errors when data is collected on instruments with limited mass resolution. The confirmation of the sequence derived from mass spectrum is best achieved by chemical synthesis and comparison of fragmentation patterns of the natural and synthetic peptides. This approach is illustrated in the determination of the sequence of a new class of conotoxins isolated from the venom of Conus araneosus. Conotoxins are particularly attractive targets for ‘de novo’ mass spectral sequencing because of the high degree of posttranslational modifications in these peptides. In this study, a two-disulfide bond containing conotoxin has been sequenced by mass spectrometry. A distinction between two possible sequences of the peptide Ar1312 has been achieved by comparing the MS/MS pattern for the natural and synthetic sequences. Confirmation of the derived sequence RCCGYKMCHOC and disulfide bond pattern suggest that Ar1312 is a χ-conotoxin, a class known to inhibit the norepinephrine transporter (NET).
Methods and tools
- Conus araneosus chi-conotoxin Ar1312: De novo sequences Ar1312, a two-disulfide conotoxin from Conus araneosus venom, by MALDI MS/MS, and resolves between two candidate sequences by comparing the natural and synthetic peptides’ fragmentation, placing it in the chi-conotoxin class.