High fidelity approach for proteomic scale enrichment and identification of N-termini

A novel workflow was designed for the large-scale identification of protein N-terminal sequences. The workflow started with converting lysine to homoarginine, followed by reaction with sulfonation of N-termini by 4-sulfophenyl isothiocyanate (SPITC). Upon trypsin digestion, the SPITC modified N-terminal peptides were enriched by electrostatic repulsion hydrophilic interaction (ERLIC) chromatography in which the internal and C-terminal peptides eluted at the void volume, and the SPITC peptides were retained in the column due to the hydrophilicity and electrostatic attraction of the sulfonyl group to the stationary phase. Upon higher-energy collisional induced dissociation (HCD) SPITC peptides in ESI generated predominately y-type ion series; such simplification of spectra enables the identification of N-termini with high confidence. The appearance of b1 + SPITC product ions upon HCD further boosts the confidence for N-terminal identifications. This method was applied to an Escherichia coli cell lysate, thus allowing the identification of 358 high confidence N-terminal peptides representing 274 distinct E. coli proteins as certain proteins were found to have multiple N-terminal peptides due to cleavage from various cellular enzymes. Confidence in N-terminal assignments is further heightened since 224 of the unique proteins identified from N-terminal peptides were also identified in the analysis of flow-through fractions.