Ultrafast microwave-assisted in-tip digestion of proteins.
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.
Hahn
Hans W
HW
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria.
Rainer
Matthias
M
Ringer
Thomas
T
Huck
Christian W
CW
Bonn
Günther K
GK
D000818:Animals; D001331:Automation; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D008872:Microwaves; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012371:Robotics; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
D002364:Caseins; D004800:Enzymes, Immobilized; D009211:Myoglobin; D011506:Proteins; D012710:Serum Albumin, Bovine; D014357:Trypsin