Molecular Scanner Development

In order to increase the throughput of protein identification and characterisation in proteome studies, we investigated three 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 immobilised trypsin which are intercalated between the gel and a PVDF collecting membrane. During electrotransfer, some digestion of the transferred proteins occurs, although poorly for basic and/or high molecular weight (MW) proteins. The second method is based on “In-Gel” digestion of all proteins in parallel, and termed “Parallel In-Gel Digestion” (PIGD) to denote this fact. The PIGD led to more efficient digestion of basic and high MW proteins (> 40 kDa) but suffered from a major drawback: loss of resolution for low molecular weight polypeptides (< 60 kDa) through diffusion during the digestion process. The third method examined was the combination of PIGD and OSDT procedures. This combination call “Double Parallel Digestion” (DPD), led to greatly improved digestion of high molecular weight and basic proteins without losses of low MW polypeptides. Peptides liberated during transblotting of proteins through the immobilised trypsin membrane were trapped on a PVDF membrane and identified by mass spectrometry in scanning mode (see Chapter 4 (Binz, Wilkins et al., 1999)).