Journal
ELECTROPHORESIS
Volume 31, Issue 22, Pages 3703-3710Publisher
WILEY-BLACKWELL
DOI: 10.1002/elps.201000317
Keywords
Microfluidics; MS; Packed column; SPE; Trypsin digestion
Funding
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- PE/Sciex
- Genome Prairie Canada
- National Institute for Nanotechnology (NINT-NRC)
- Defence Research Development Canada-Suffield
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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 x 30 mu m channels for CE, and two serially connected 150 mu m deep, 800 mu m wide channels packed with 40 to 60 mu m 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 mu g/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.
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