De Novo Sequencing of Tryptic Phosphopeptides Using Matrix-Assisted Laser Desorption/Ionization Based Tandem Mass Spectrometry with Hydrogen Atom Attachment
peer-reviewed · Analytical Chemistry · 2018
| Date | 2018-02-20 |
| Type | peer-reviewed |
| Venue | Analytical Chemistry |
| Publisher | American Chemical Society (ACS) |
| Contribution | algorithm |
| DOI | 10.1021/acs.analchem.7b04635 |
| Citations (OpenAlex) | 17 |
| Venue 2-year citedness | 7.10 |
Abstract
Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS/MS) with radical-based fragmentation techniques has proven to be a promising method for phosphoproteomic applications, owing to its ability to determine phosphorylation sites on proteins. The radical-induced fragmentation technique involves the attachment or abstraction of hydrogen to peptides in an ion trap mass spectrometer, in a process called hydrogen attachment/abstraction dissociation (HAD), which has only been recently developed. In the present investigation, we have analyzed model phosphopeptides and phosphoprotein digests using HAD-MS/MS, combined with matrix-assisted laser desorption/ionization (MALDI), in order to demonstrate the usefulness of the HAD-MS/MS-based analytical method. The tryptic peptides were categorized as arginine- and lysine-terminated peptides, and MALDI HAD-MS/MS is found to facilitate the sequencing of arginine-terminated tryptic peptides, because of the selective observation of C-terminal side fragment ions. In contrast, MALDI HAD-MS/MS of lysine-terminated tryptic peptides produced both N- and C-terminal side fragments, such that the mass spectra were complex. The guanidination of peptide converted lysine into homoarginine, which facilitated the interpretation of MALDI HAD-MS/MS mass spectra. The present method was useful for de novo sequencing of tryptic phosphopeptides.
Methods and tools
- Hydrogen atom attachment phosphopeptide sequencing: Radical fragmentation by hydrogen atom attachment on a MALDI instrument, which keeps the labile phosphate in place while cleaving the backbone, so tryptic phosphopeptides can be sequenced de novo and their phosphosites located.