ac8010704_si_004.xls (351.5 kB)
Download file

Versatile Online−Offline Engine for Automated Acquisition of High-Resolution Tandem Mass Spectra

Download (351.5 kB)
posted on 01.11.2008, 00:00 authored by Craig D. Wenger, Michael T. Boyne, Jonathan T. Ferguson, Dana E. Robinson, Neil L. Kelleher
For automated production of tandem mass spectrometric data for proteins and peptides >3 kDa at >50 000 resolution, a dual online−offline approach is presented here that improves upon standard liquid chromatography−tandem mass spectrometry (LC−MS/MS) strategies. An integrated hardware and software infrastructure analyzes online LC−MS data and intelligently determines which targets to interrogate offline using a posteriori knowledge such as prior observation, identification, and degree of characterization. This platform represents a way to implement accurate mass inclusion and exclusion lists in the context of a proteome project, automating collection of high-resolution MS/MS data that cannot currently be acquired on a chromatographic time scale at equivalent spectral quality. For intact proteins from an acid extract of human nuclei fractionated by reversed-phase liquid chromatography (RPLC), the automated offline system generated 57 successful identifications of protein forms arising from 30 distinct genes, a substantial improvement over online LC−MS/MS using the same 12 T LTQ FT Ultra instrument. Analysis of human nuclei subjected to a shotgun Lys-C digest using the same RPLC/automated offline sampling identified 147 unique peptides containing 29 co- and post-translational modifications. Expectation values ranged from 10−5 to 10−99, allowing routine multiplexed identifications.