Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix‐assisted laser desorption/ionization de novo sequencing
peer-reviewed · Rapid Communications in Mass Spectrometry · 2016
| Date | 2016-07-30 |
| Type | peer-reviewed |
| Venue | Rapid Communications in Mass Spectrometry |
| Publisher | Wiley |
| Contribution | adjacent |
| DOI | 10.1002/rcm.7594 |
| Citations (OpenAlex) | 6 |
| Venue 2-year citedness | 1.80 |
Abstract
Rationale One of the most challenging tasks of proteomics is peptide de novo sequencing. 4‐Sulfophenyl isothiocyanate (SPITC) peptide derivatization enables acquisition of high‐quality tandem mass spectra (MS/MS) for de novo sequencing, but unwanted non‐specific reactions and reduced mass spectra (MS) signal intensities still represent the obstacles in high‐throughput de novo sequencing. Methods We developed a SPITC peptide derivatization procedure under acidic conditions (pH ≤5). Derivatized peptides were analyzed by matrix‐assisted laser desorption/ionization (MALDI‐MS) in negative ion mode followed by MS/MS in positive ion mode. A de novo sequencing tool, named DUST, adjusted to SPITC chemistry, was designed for successful high‐throughput peptide de novo sequencing. This high‐throughput peptide de novo sequencing was tested on Fusarium delphinoides , an organism with an uncharacterized genome. Results The SPITC derivatization procedure under acidic conditions produced a significantly improved MS dataset in comparison to commonly used derivatization under basic conditions. Signal intensities were 6 to 10 times greater and the over‐sulfonation effect measured on lysine‐containing peptides was significantly decreased. Furthermore, development of a novel DUST algorithm enabled automated de novo sequencing with the calculated accuracy of 70.6%. Conclusions The SPITC derivatization and de novo sequencing approach outlined here provides a reliable method for high‐throughput peptide de novo sequencing. High‐throughput peptide de novo sequencing enabled protein mutation identification and identification of proteins from organisms with non‐sequenced genomes. Copyright © 2016 John Wiley & Sons, Ltd.
Methods and tools
- DUST and SPITC CAF MALDI de novo sequencing (Zagreb): SPITC derivatization of tryptic peptides, done at acidic pH to avoid side reactions, with chemically activated fragmentation on MALDI and the DUST de novo tool tuned to SPITC chemistry.
Cites (4)
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- De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue‐green alga Aphanizomenon flos‐aquae (2009) crossref
- Highly informative proteome analysis by combining improved N ‐terminal sulfonation for de novo peptide sequencing and online capillary reverse‐phase liquid chromatography/tandem mass spectrometry (2004) crossref
- Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry (2000) crossref