Toward a Clinical Molecular Scanner for Proteome Research: Parallel Protein Chemical Processing before and during Western Blot

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• 作者：Willy V. Bienvenut ; Jean-Charles Sanchez ; Abderrahim Karmime ; Vé ; ronique Rouge ; Keith Rose ; Pierre-Alain Binz ; and Denis F. Hochstrasser
• 刊名：Analytical Chemistry
• 出版年：1999
• 出版时间：November 1, 1999
• 年：1999
• 卷：71
• 期：21
• 页码：4800 - 4807
• 全文大小：310K
• 年卷期：v.71,no.21(November 1, 1999)
• ISSN：1520-6882

文摘

To increase the throughput of protein identification andcharacterization in proteome studies, we investigatedthree methods of performing protein digestion in parallel.The first, which we term "one-step digestion-transfer"(OSDT), is based on protein digestion during the transblotting process. It involves the use of membranes containing immobilized trypsin which are intercalated between the gel and a PVDF collecting membrane. Duringelectrotransfer, some digestion of the transferred proteinsoccurs, although poorly for basic and/or high molecularweight proteins. The second method is based on "in-gel"digestion of all proteins in parallel and termed "parallelin-gel digestion" (PIGD) to denote this fact. The PIGD ledto more efficient digestion of basic and high molecularweight proteins (>40 000) but suffered from a majordrawback: loss of resolution for low molecular weightpolypeptides (<60 000) through diffusion during thedigestion process. The third method examined was thecombination of PIGD and OSDT procedures. This combination, called "double parallel digestion" (DPD), led togreatly improved digestion of high molecular weight andbasic proteins without losses of low molecular weightpolypeptides. Peptides liberated during transblotting ofproteins through the immobilized trypsin membrane weretrapped on a PVDF membrane and identified by massspectrometry in scanning mode (Binz et al. Anal. Chem.1999, 71, 4981-4988).