Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS.
An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 microm wide, 100 microm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 microL/min, producing a reaction time of approximately 24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, bovine serum albumin (BSA), myoglobin, and phosphorylase b, respectively.
Lee
Jeonghoon
J
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70802, USA.
Musyimi
Harrison K
HK
Soper
Steven A
SA
Murray
Kermit K
KK
D000818:Animals; D019149:Bioreactors; D002417:Cattle; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D019904:Polymethyl Methacrylate; D040901:Proteomics / Q000379:methods; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods; D014357:Trypsin / Q000737:chemistry
D019904:Polymethyl Methacrylate; D014357:Trypsin