A method for N-terminal de novo sequencing of N α -blocked proteins by mass spectrometry
peer-reviewed · The Analyst · 2010
| Date | 2010-10-08 |
| Type | peer-reviewed |
| Venue | The Analyst |
| Publisher | Royal Society of Chemistry (RSC) |
| Contribution | algorithm |
| DOI | 10.1039/c0an00384k |
| Citations (OpenAlex) | 20 |
Abstract
A method for de novo sequencing of N(α)-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of N(α)-blocked protein, recovery of N-terminal peptide by depletion of non-N-terminal peptides from the digest pool, and selective derivatization of a C-terminal α-carboxyl group of isolated N-terminal peptide. The C-terminal α-carboxyl group of the N-terminal peptide was selectively derivatized with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine), according to oxazolone chemistry. The reagent TMPP-propylamine was designed to facilitate sequence analysis with MALDI-MS by mass- and charge-tagging. All of the identities and N-terminal sequences of two N(α)-acetylated proteins (rabbit phosphorylase b and bovine calmodulin) and human orexin A, which has pyroglutamic acid at the N-terminus, were successfully analyzed by allowing for the y-type ions almost exclusively.
Methods and tools
- TMPP N-terminal peptide de novo sequencing: Tags peptide N-termini with TMPP-Ac-OSu after metalloendopeptidase digestion and isolates them, giving MALDI spectra of N-terminal peptides clean enough to sequence de novo.
Cites (4)
- Selective isolation of N‐terminal peptides from proteins and their de novo sequencing by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry without regard to unblocking or blocking of N‐terminal amino acids (2008) crossref
- A method for N-terminal de novo sequence analysis of proteins by matrix-assisted laser desorption/ionization mass spectrometry (2008) crossref
- An improved method for de novo sequencing of arginine‐containing, N α ‐tris(2,4,6‐trimethoxyphenyl)phosphonium‐acetylated peptides (2008) crossref
- De novo sequencing of proteolytic peptides by a combination of C-terminal derivatization and nano-electrospray/ collision-induced dissociation mass spectrometry (2000) crossref