De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature
peer-reviewed · Analytical and Bioanalytical Chemistry · 2012
| Date | 2012-02-01 |
| Type | peer-reviewed |
| Venue | Analytical and Bioanalytical Chemistry |
| Publisher | Springer Science and Business Media LLC |
| Contribution | algorithm |
| DOI | 10.1007/s00216-011-5642-7 |
| Citations (OpenAlex) | 14 |
Abstract
De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.
Methods and tools
- Br-signature N-terminal derivatization: A MALDI signal-enhancing N-terminal derivatization carrying a bromine isotope signature, so the tagged N-terminal peptide is unmistakable in the spectrum. Used to read N-terminal processing such as methionine or signal-peptide removal, and to annotate uncharacterized proteins.
Cites (8)
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