Integrating accelerated tryptic digestion into proteomics workflows.
An accelerated protein digestion procedure is described that features a microscale trypsin cartridge operated under aqueous-organic conditions. High sequence coverage digestions obtained in seconds with small amounts of enzyme are possible with the approach, which also supports online integration of digestion with reversed-phase protein separation. The construction and operation of effective digestor cartridges for rapid sample processing are described. For workflows involving chromatographic protein separation an easily assembled fluidic system is presented, which inserts the digestion step after column-based separation. Successful integration requires dynamic effluent titration immediately prior to transmission through the digestor. This is achieved through the co-ordination of the column gradient system with an inverse gradient system to produce steady pH and organic solvent levels. System assembly and operation sufficient for achieving digestion and identification of subnanogram levels of protein are described.
Slysz
Gordon W
GW
Department of Chemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
Schriemer
David C
DC
D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
D010455:Peptides; D014357:Trypsin; D002256:Carbonic Anhydrases