Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics.
J Proteome Res
; 7(3): 1007-11, 2008 Mar.
Article
em En
| MEDLINE
| ID: mdl-18247544
ABSTRACT
Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
Cromatografia Líquida
/
Proteômica
Tipo de estudo:
Evaluation_studies
Limite:
Animals
Idioma:
En
Revista:
J Proteome Res
Ano de publicação:
2008
Tipo de documento:
Article