Coupling a detergent lysis/cleanup methodology with intact protein fractionation for enhanced proteome characterization.
J Proteome Res
; 11(12): 6008-18, 2012 Dec 07.
Article
em En
| MEDLINE
| ID: mdl-23126408
ABSTRACT
The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples ( Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Microbiologia da Água
/
Proteoma
/
Proteômica
/
Detergentes
/
Fracionamento Químico
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos