Subfemtomole MS and MS/MS Peptide Sequence Analysis Using Nano-HPLC Micro-ESI Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Subfemtomole peptide sequence analysis has been achieved using microcapillary HPLC columns, with integrated nanoelectrospray emitters, coupled directly to a Fourier transform ion cyclotron resonance mass spectrometer. Accurate mass (±0.010 Da) peptide maps are generated from a standard six-protein digest mixture, whose principle components span a concentration dynamic range of 1000:1. Iterative searches against ∼189 000 entries in the OWL database readily identify each protein, with high sequence coverage (20−60%), from as little as 10 amol loaded on-column. In addition, a simple variable-flow HPLC apparatus provides for on-line tandem mass spectrometric analysis of tryptic peptides at the 400-amol level. MS/MS data are searched against ∼280 000 entries in a nonredundant protein database using SEQUEST. Accurate precursor and product ion mass information readily identifies primary amino acid sequences differing by asparagine vs aspartic acid (Δm = 0.98 Da) and glutamine vs lysine (Δm = 0.036 Da).