Ultrafast Microwave-Assisted In-Tip Digestion of Proteins

J Proteome Res. 2009 Sep;8(9):4225-30. doi: 10.1021/pr900188x.

Abstract

Trypsin was immobilized on glycidylmethacrylate-co-divinylbenzene (GMA/DVB) polymerized in pipet tips for online enzymatic digestion of proteins. The major advantages of in-tip digestion are easy handling and small sample amount required for analysis. Microwave-assisted digestion was applied for highly efficient and time saving proteolysis. Adaption to an automated robotic system allowed fast and reproducible sample treatment. Investigations with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography coupled with electrospray-ionization mass spectrometry (LC-ESI/MS) attested high sequence coverages (SCs) for the three standard proteins, myoglobin (Myo, 89%), bovine serum albumin (BSA, 78%) and alpha-casein (alpha-Cas, 83%). Compared to commercially available trypsin tips, clear predominance concerning the digestion performance was achieved. Storageability was tested over a period of several weeks and results showed only little decrease (<5%) of protein sequence coverages. The application of microwave-assisted in-tip digestion (2 min) with full automation by a robotic system allows high-throughput analysis (96 samples within 80 min) and highly effective proteolysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Automation
  • Caseins / chemistry
  • Caseins / metabolism
  • Cattle
  • Chromatography, Liquid / methods
  • Enzyme Stability
  • Enzymes, Immobilized / chemistry
  • Enzymes, Immobilized / metabolism*
  • Microwaves*
  • Myoglobin / chemistry
  • Myoglobin / metabolism
  • Proteins / chemistry
  • Proteins / metabolism*
  • Proteomics / methods*
  • Reproducibility of Results
  • Robotics
  • Serum Albumin, Bovine / chemistry
  • Serum Albumin, Bovine / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
  • Time Factors
  • Trypsin / chemistry
  • Trypsin / metabolism*

Substances

  • Caseins
  • Enzymes, Immobilized
  • Myoglobin
  • Proteins
  • Serum Albumin, Bovine
  • Trypsin