Toward a clinical molecular scanner for proteome research: Parallel protein chemical processing before and during western blot

To increase the throughput of protein identification and characterization i n proteome studies, we investigated three methods of performing protein dig estion 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 inte rcalated between the gel and a PVDF collecting membrane. During electrotran sfer, some digestion of the transferred proteins occurs, although poorly fo r basic and/or high molecular weight proteins, The second method is based o n "in-gel" digestion of all proteins in parallel and termed "parallel in-ge l digestion" (PIGD) to denote this fact. The PIGD led to more efficient dig estion of basic and high molecular weight proteins (>40 000) but suffered f rom a major drawback: loss of resolution for low molecular weight polypepti des (>60 000) through diffusion during the digestion process. The third met hod examined was the combination of PIGD and OSDT procedures. This combinat ion, called "double parallel digestion" (DPD), led to greatly improved dige stion of high molecular weight and basic proteins without losses of low mol ecular weight polypeptides, Peptides liberated during transblotting of prot eins through the immobilized trypsin membrane were trapped on a PVDF membra ne and identified by mass spectrometry in scanning mode.