Multifunctional protein processing chip with integrated digestion, solid-phase extraction, separation and electrospray.
We describe a microfluidic device in which integrated tryptic digestion, SPE, CE separation and electrospray ionization for MS are performed. The chip comprised of 10 × 30 μm channels for CE, and two serially connected 150 μm deep, 800 μm wide channels packed with 40 to 60 μm diameter beads, loaded with either immobilized trypsin, reversed-phase packing or both. On-chip digestion of cytochrome c using the trypsin bed showed complete consumption of the protein in 3 min, in contrast to the 2 h required for conventional solution phase tryptic digestion. SPE of 0.25 μg/mL solutions of the peptides leu-enkephalin, angiotensin II and LHRH gave concentration enhancements in the range of 4.4-12, for a ten times nominal volume ratio. A 100 nM cytochrome c sample concentrated 13.3 times on-chip gave a sequence coverage of 85.6%, with recovery values ranging from 41.2 to 106%. The same sample run without SPE showed only five fragment peaks and a sequence coverage of 41.3%. When both on-chip digestion and SPE (13.3 volume ratio concentration enhancement) were performed on 200 nM cytochrome c samples, a sequence coverage of 76.0% and recovery values of 21-105% were observed. Performing on-chip digestion alone on the same sample gave only one significant fragment peak. The above digestion/peptide concentration step was compared to on-chip protein concentration by SPE followed by on-chip digestion with solution phase trypsin. Both procedures gave similar recovery results; however, much larger trypsin autodigestion interference in the latter approach was apparent.
Department of Chemistry, University of Alberta, Edmonton, AB, Canada.
Can
C
Wang
Abebaw B
AB
Jemere
D Jed
DJ
Harrison
D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D046209:Electrophoresis, Microchip / Q000295:instrumentation*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D052617:Solid Phase Microextraction / Q000295:instrumentation*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism*
D045304:Cytochromes c; D014357:Trypsin