Specific isolation of N‐terminal fragments from proteins and their high‐fidelity de novo sequencing
peer-reviewed · Rapid Communications in Mass Spectrometry · 2007
| Date | 2007-10-30 |
| Type | peer-reviewed |
| Venue | Rapid Communications in Mass Spectrometry |
| Publisher | Wiley |
| Contribution | algorithm |
| DOI | 10.1002/rcm.3215 |
| Citations (OpenAlex) | 21 |
| Venue 2-year citedness | 1.80 |
Abstract
A new method to determine N-terminal amino acid sequences of multiple proteins at low pmol level by a parallel processing has been developed. The method contains the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling with sulfosucccimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate(sulfo-NHS-SS-biotin) to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease; (4) specific isolation of N-terminal fragments of proteins by affinity capture using the biotin-avidin system; (5) de novo sequence analysis of peptides by MALDI-TOF-/MALDI-TOF-PSD mass spectrometry with effective utilization of the CAF (chemically assisted fragmentation) method.1 This method is also effective for N-terminal sequencing of each protein in a mixture of several proteins, and for sequencing components of a multiprotein complex. It is expected to become an essential proteomics tool for identifying proteins, especially when used in combination with a C-terminal sequencing method.
Methods and tools
- N-terminal fragment isolation de novo sequencing: Isolates protein N-terminal peptides specifically, through reduction, S-alkylation, guanidination and a cleavable biotin coupling, so several proteins can be N-terminally sequenced in parallel at low picomole level.