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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...
Protein identification is becoming a complement to the available fully sequenced genomes. To meet the challenge, newly developed techniques for high throughput protein identification using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and peptide mass fingerprint are needed. Two years ago, a parallel protein digestion process was proposed. It provided a collecting polyvinylidene...
Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today’s commonly used high throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification...
The molecular scanner combines protein separation using gel electrophoresis with peptide mass fingerprinting (PMF) techniques to identify proteins in a highly automated manner. Proteins separated in a 2-dimensional polyacrylamide gel (2D-PAGE) are digested ‘in parallel’ and transferred onto a membrane keeping their relative positions. The membrane is then sprayed with a matrix and inserted into a...
Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 15 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific...
Although PMF is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the...
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