期刊
ANALYTICAL CHEMISTRY
卷 80, 期 21, 页码 8055-8063出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac8010704
关键词
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资金
- National Institutes of Health [GM 067193-06]
- Packard Foundation
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.
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