Autopilot: An Online Data Acquisition Control Systemfor the Enhanced High-Throughput Characterization of Intact Proteins

摘要

The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the softwarecombines fragmentation events of the same precursor with previouslyobtained fragments to achieve improved characterization of the targetform by an average of 42 orders of magnitude in confidence. When HCDfragmentation optimization was applied to intact proteins ions, therewas an 18.5 order of magnitude gain in confidence. These improvedmetrics set the stage for increased proteome coverage and characterizationof higher order organisms in the future for sharply improved controlover MS instruments in a project- and lab-wide context.