Precursor ion independent algorithm for top-down shotgun proteomics

Abstract

We present a precursor ion independent top-down algorithm (PIITA) for use in automated assignment of protein identifications from tandem mass spectra of whole proteins. To acquire the data, we utilize data-dependent acquisition to select protein precursor ions eluting from a C4-based HPLC column for collision induced dissociation in the linear ion trap of an LTQ-Orbitrap mass spectrometer. Gas-phase fractionation is used to increase the number of acquired tandem mass spectra, all of which are recorded in the Orbitrap mass analyzer. To identify proteins, the PIITA algorithm compares deconvoluted, deisotoped, observed tandem mass spectra to all possible theoretical tandem mass spectra for each protein in a genomic sequence database without regard for measured parent ion mass. Only after a protein is identified, is any difference in measured and theoretical precursor mass used to identify and locate post-translation modifications. We demonstrate the application of PIITA to data generated via our wet-lab approach on a Salmonella typhimurium outer membrane extract and compare these results to bottom-up analysis. From these data, we identify 154 proteins by top-down analysis, 73 of which were not identified in a parallel bottom-up analysis. We also identify 201 unique isoforms of these 154 proteins at a false discovery rate (FDR) of <1%.