Peptide de Novo Sequencing Using 157 nm Photodissociation in a Tandem Time-of-Flight Mass Spectrometer
peer-reviewed · Analytical Chemistry · 2010
| Date | 2010-02-01 |
| Type | peer-reviewed |
| Venue | Analytical Chemistry |
| Publisher | American Chemical Society (ACS) |
| Contribution | algorithm |
| DOI | 10.1021/ac902050y |
| Citations (OpenAlex) | 38 |
| Venue 2-year citedness | 6.29 |
Abstract
It has previously been shown that photodissociation of tryptic peptide ions with 157 nm light in a matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF) mass spectrometer generates an abundance of x-type ions. A peptide de novo sequencing algorithm has now been developed to interpret these data. By combination of photodissociation and postsource decay (PSD) spectra, the algorithm identifies x-type ions and derives peptide sequences. The confidence of amino acid assignments is evaluated by observing complementary y-, v-, and w-type ions that provide additional constraints to sequence identification. In the analysis of 31 tryptic peptides from 4 model proteins, the algorithm identified 322 (or 90.7%) of the 355 amino acids and made only 3 incorrect assignments. The other 30 amino acids were not identified because specific needed x-type ions were not detected. Based on the observation of v- and w-type ions, 45 of 50 detected leucine and isoleucine residues were successfully distinguished and there was only one mistake. The remaining four residues were not distinguished because the corresponding v- and w-type ions were not detected. These de novo sequencing results translated into successful identification of proteins through homology searches. To evaluate the robustness of the present sequencing approach, a collection of 266 tryptic peptides from 23 model proteins were analyzed and then sequenced. A total of 167 peptides yielded sequence tags of 5 or more residues. In 5 peptides, 1 or 2 residues were incorrectly assigned.
Methods and tools
- 157 nm photodissociation de novo sequencing: De novo sequencing algorithm written for 157 nm photodissociation spectra, which yield clean x-ion series rather than the mixed b/y ions of CID. Reports per-residue accuracy and distinguishes leucine from isoleucine, which equal-mass residues normally make impossible.