Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C1389183 (autodigestion)
 317 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a gel electrophoresis system that can concentrate proteins from spots cut out of up to 50 two-dimensional electrophoresis gels. During protein concentration, SDS is substituted with a non-ionic detergent (octyl beta-glucopyranoside) which allows digestion and MS analysis of the protein directly extracted from the gel without fixation or staining. The system avoids the problems associated with the digestion of dilute protein in multiple bands by (a) greatly reducing the gel volume for digestion and thus the amount of protease required, hence lowering contamination by autodigestion products, (b) reducing the volume of solvent required for extraction of protein from the gel, thus minimising loss of material to container surfaces, and (c) removing SDS which interferes with subsequent MS or HPLC analysis. The efficiency of protein recovery ranges between an average of 80% for proteins from silver stained two-dimensional gels to 90% for fluorescence and Coomassie-blue-stained gels. The method is compatible with MS analysis of very low amounts of protein from any staining system, but appears not to be useful for Edman sequencing of silver-stained or fluorescent-stained proteins since the amount of N-terminal blockage appears to increase as the amount of protein isolated from the two-dimensional gel decreases.
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PMID:Concentration of, and SDS removal from proteins isolated from multiple two-dimensional electrophoresis gels. 920 22

The routine proteolysis of proteins is performed in solution, but it suffers from drawbacks such as long incubation time, enzyme autodigestion, and non-reusability. Therefore we here demonstrated that trypsin could be immobilized on silver wire modified by atom transfer radical polymerization to prepare a new kind of enzyme immobilized reactor. The digestion efficiency, repeatability and recovery of the silver wire-trypsin reactor (SW-Trypsin) were evaluated by mass spectrometry (MS) analysis. Highly efficient digestion was achieved by using SW-Trypsin within only 20 min. Standard protein could be almost completely digested with sequence coverage up to 93%, which is higher than that of 79% sequence coverage obtained by in-solution digestion for 16 h. Bovine serum albumin (BSA) was digested eight times within a month by using the SW-Trypsin. The results of sequence coverage were between 89% to 97%, with an average sequence coverage of 94%, which showed that SW-Trypsin had good stability. In addition, the recovery test by using myoglobin (MYO) showed that the recovery rate was 87.67%. At last, the extract from Tengchong thermophilic bacteria was digested by SW-Trypsin in 20 min and in-solution trypsin in 16 h. The results of sequence coverages and the number of identified proteins were similar. Moreover, the ratio of the number of peptides with zero missed cleavages to the number of all identified peptides by using SW-Trypsin was higher than that by in-solution digestion. Also, the SW-Trypsin was easily removed from the digestion solution. Good performances of SW-Trypsin implied that it has a good prospect in the application in future proteomics research.
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PMID:[Preparation of a trypsin immobilized reactor on silver wire modified by atom transfer radical polymer and its application in proteome identification]. 2389 35