1. Versatile Online--Offline Engine for Automated Acquisition of High-Resolution Tandem Mass Spectra.
- Author
-
Wenger, Craig D., Boyne, II, Michael T., Ferguson, Jonathan T., Robinson, Dana E., and Kelleher, Neil L.
- Subjects
- *
TANDEM mass spectrometry , *CHEMINFORMATICS , *PROTEOMICS , *ANALYTICAL chemistry techniques , *LIQUID chromatography , *AUTOMATIC data collection systems , *POST-translational modification , *SAMPLING (Process) - Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF