P164-M Optimization of the Gel Shredder: A Centrifugal Device for Automated In-gel Digestion with Improved Peptide Recovery.

摘要

Proteomic workflow frequently involves gel separations. Poor peptide recovery from in-gel digestion procedures is at large influenced by the properties of polyacrylamide gel, which restricts access of proteolytic enzymes to the protein substrate as well as prevents complete extraction of certain peptides. We have designed an optimized centrifugal device which allows integrated washing, destaining and shredding of gel bands into uniform blocks of controlled size, roughly 100 μm, prior to the enzymatic digestion. Such treatment increases the surface area of gel pieces and allows the enzymes and solvent to better penetrate the gel lattice. The performance of such novel device has been evaluated using several gel-separated model proteins pre-labeled with thiol-specific multiplexed stable isotope tags, followed by relative quantification by a MALDI-TOF/TOF mass spectrometer. The performance of the new device, called the Gel Shredder, has been compared to standard in-gel digestion protocols. It has been shown that an improvement in peptide recovery can be reproducibly obtained by using the Gel Shredder devices. It has been determined that the application of the Gel Shredder is particularly useful for recoveries of high molecular weight hydrophobic peptides, presumably due to their higher affinity to the polyacrylamide gel lattice. Gel Shredder also allows to save time usually spent on gel band manipulation during in-gel digestion experiments.