Rheostatic control of tryptic digestion in a microscale fluidic system.
Integrated fluidic systems that unite bottom-up and top-down proteomic approaches have the potential to deliver complete protein characterization. To circumvent fraction collection, as is conducted in current blended approaches, a technique to regulate digestion efficiency in a flow-through system is required. The present study examined the concept of regulating tryptic digestion in an immobilized enzyme reactor (IMER), incorporating mixed solvent systems for digestion acceleration. Using ovalbumin, cytochrome c, and myoglobin as protein standards, we demonstrate that tryptic digestion can be efficiently regulated between complete digestion and no digestion extremes by oscillating between 45 and 0% acetonitrile in the fluid stream. Solvent composition was tuned using programmable solvent waveforms in a closed system consisting of the IMER, a sample delivery stream, a dual gradient pumping system and a mass spectrometer. Operation in this rheostatic digestion mode provides access to novel peptide mass maps (due to substrate unfolding hysteresis) as well as the intact protein, in a reproducible and stable fashion. Although cycle times were on the order of 90 s for testing purposes, we show that regulated digestion is sufficiently rapid to be limited by solvent switching efficiency and kinetics of substrate unfolding/folding. Thus, regulated digestion should be useful in blending bottom-up and top-down proteomics in a single closed fluidic system.
Percy
Andrew J
AJ
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Schriemer
David C
DC
D019149:Bioreactors / Q000382:microbiology; D002851:Chromatography, High Pressure Liquid; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012997:Solvents / Q000737:chemistry; D014357:Trypsin
D010455:Peptides; D012997:Solvents; D045304:Cytochromes c; D014357:Trypsin