Centrifugal methods and devices for rapid in-gel digestion of proteins.
Modern proteomic research frequently relies upon separation of proteins in a polyacrylamide gel matrix followed by in-gel enzymatic digestion and extraction of peptides for subsequent analysis by MS. In this work, we propose a novel semi-automated method of mechanical processing of gel bands by passing these bands through a specially designed centrifugal device termed a Gel Shredder prior to digestion and extraction of peptides. Such a device allows integrated washing, destaining and shredding of gel bands into uniform blocks of controlled size, approximately 150-300 microm, prior to the enzymatic digestion and extraction of peptides. Shredding into uniform blocks increases the surface area of the gel pieces and promotes improved gel rehydration, allowing improved diffusion of the proteolytic enzymes and solvent into the gel lattice. We demonstrate that the new method substantially reduces the time spent on tedious manual handling of gel bands, while minimizing the risk of sample contamination. The performance of the Gel Shredder has been compared with a conventional in-gel digestion protocol using several standard proteins and a complex proteomic sample in terms of relative quantitation by either MALDI-TOF/TOF or nanoLC-ESI IT-Fourier transformation ion cyclotron resonance MS. It is shown that significant time savings and improved peptide recovery can be obtained for many proteins using the Gel Shredder compared with the traditional in-gel digestion protocol.
Lazarev
Alexander V
AV
Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, USA. alazarev@pressurebiosciences.com
Rejtar
Tomas
T
Dai
Shujia
S
Karger
Barry L
BL
D000273:Adipose Tissue / Q000737:chemistry; D000818:Animals; D002498:Centrifugation; D002853:Chromatography, Liquid; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D004867:Equipment Design; D013058:Mass Spectrometry; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin