A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome.
J Am Soc Mass Spectrom
; 20(12): 2183-91, 2009 Dec.
Article
en En
| MEDLINE
| ID: mdl-19747844
ABSTRACT
For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Electroforesis en Gel Bidimensional
/
Biomarcadores de Tumor
/
Espectroscopía Infrarroja por Transformada de Fourier
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Proteoma
/
Fraccionamiento Químico
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Proteínas de Neoplasias
Tipo de estudio:
Evaluation_studies
Límite:
Humans
Idioma:
En
Revista:
J Am Soc Mass Spectrom
Año:
2009
Tipo del documento:
Article
País de afiliación:
Estados Unidos