Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

  • Jeonghoon Lee
  • Harrison K. Musyimi
  • Steven A. Soper
  • Kermit K. Murray
Article

Abstract

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 µm wide, 100 µm 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 µL/min, producing a reaction time of ∼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.

Supplementary material

13361_2011_190700964_MOESM1_ESM.doc (161 kb)
Supplementary material, approximately 165 KB.

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Copyright information

© American Society for Mass Spectrometry 2008

Authors and Affiliations

  • Jeonghoon Lee
    • 1
  • Harrison K. Musyimi
    • 2
  • Steven A. Soper
    • 1
    • 3
    • 4
  • Kermit K. Murray
    • 1
  1. 1.Department of ChemistryLouisiana State UniversityBaton RougeUSA
  2. 2.National Institute on Aging/National Institutes of HealthBaltimoreUSA
  3. 3.Department of Mechanical EngineeringLouisiana State UniversityBaton RougeUSA
  4. 4.Center for BioModular Multi-Scale SystemsLouisiana State UniversityBaton RougeUSA

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