Versatile online-offline engine for automated acquisition of high-resolution tandem mass spectra.
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.
Wenger
Craig D
CD
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Boyne
Michael T
MT
Ferguson
Jonathan T
JT
Robinson
Dana E
DE
Kelleher
Neil L
NL
D000595:Amino Acid Sequence; D006367:HeLa Cells; D006801:Humans; D020407:Internet / Q000295:instrumentation*; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D040901:Proteomics; D053719:Tandem Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*
D011506:Proteins