Specific isolation of N‐terminal fragments from proteins and their high‐fidelity de novo sequencing

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 α ‐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.2, 3 Copyright © 2007 John Wiley & Sons, Ltd.