Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.
Methods
; 104: 163-9, 2016 07 15.
Article
em En
| MEDLINE
| ID: mdl-26826592
ABSTRACT
The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein 'A' on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated 'top-down' MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of 'top-down' and 'bottom-up' proteomics.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Eletroforese em Gel Bidimensional
/
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
/
Isoformas de Proteínas
/
Proteômica
Limite:
Humans
Idioma:
En
Revista:
Methods
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Estados Unidos