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

Publisher Website
Google Scholar
Abstract
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).