A method for N-terminal de novo sequencing of N-alpha-blocked proteins by mass spectrometry

A method for de novo sequencing of N-alpha-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of N-alpha-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 alpha-carboxyl group of isolated N-terminal peptide. The C-terminal alpha-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-alpha-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.