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Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.
Scheerlinck, E; Dhaenens, M; Van Soom, A; Peelman, L; De Sutter, P; Van Steendam, K; Deforce, D.
Afiliação
  • Scheerlinck E; Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
  • Dhaenens M; Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
  • Van Soom A; Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
  • Peelman L; Department of Nutrition, Genetics, and Ethology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
  • De Sutter P; Department of Reproductive Medicine, Ghent University Hospital, B-9000 Ghent, Belgium.
  • Van Steendam K; Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
  • Deforce D; Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium. Electronic address: dieter.deforce@ugent.be.
Anal Biochem ; 490: 14-9, 2015 Dec 01.
Article em En | MEDLINE | ID: mdl-26302362
Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Mapeamento de Peptídeos / Proteômica / Métodos Analíticos de Preparação de Amostras / Proteínas de Neoplasias Limite: Humans País/Região como assunto: Europa Idioma: En Revista: Anal Biochem Ano de publicação: 2015 Tipo de documento: Article País de afiliação: Bélgica País de publicação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Mapeamento de Peptídeos / Proteômica / Métodos Analíticos de Preparação de Amostras / Proteínas de Neoplasias Limite: Humans País/Região como assunto: Europa Idioma: En Revista: Anal Biochem Ano de publicação: 2015 Tipo de documento: Article País de afiliação: Bélgica País de publicação: Estados Unidos