Development of an efficient on-chip digestion system for protein analysis using MALDI-TOF MS.
A solid-phase trypsin microreactor was constructed and operated with electrokinetically-driven flow for the digestion of proteins and coupled off-line with MALDI-TOF MS. The bioreactor was fabricated from poly(methyl methacrylate), PMMA, by hot embossing using a mold master prepared by micro-milling. The solid-phase bioreactor consisted of a 4 cm long, 200 microm wide, and 50 microm deep microfluidic channel that was populated with an array of 50 microm diameter micropost structures with a 50 microm inter-post spacing. The bioreactor was prepared by covalently attaching the proteolytic enzyme, trypsin, to the UV-modified surface of the PMMA microstructures using the appropriate coupling reagents. The performance of the system was evaluated using a set of proteins. The bioreactor provided efficient digestion of cytochrome c at a field strength of 375 V/cm, producing a reaction time of approximately 20 s to produce 97% sequence coverage for protein identification. Bovine serum albumin (BSA), phosphorylase b, and beta-casein were also assessed and the sequence coverages were 46, 63, and 79%, respectively, using the same reactor residence time. Furthermore, Escherichia coli was used as a model to demonstrate the feasibility of fingerprint analysis for intact cells using this solid-phase bioreactor.
Lee
Jeonghoon
J
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA.
Soper
Steven A
SA
Murray
Kermit K
KK
D055664:Electrochemical Techniques; D056656:Lab-On-A-Chip Devices; D019904:Polymethyl Methacrylate / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods*
D011506:Proteins; D019904:Polymethyl Methacrylate