Versatile online-offline engine for automated acquisition of high-resolution tandem mass spectra.
Anal Chem
; 80(21): 8055-63, 2008 Nov 01.
Article
em En
| MEDLINE
| ID: mdl-18841935
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.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Internet
/
Espectrometria de Massas em Tandem
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Estados Unidos