Trypsin-linked copolymer MALDI chips for fast protein identification.
For the first time, trypsin-linked copolymer poly(methyl methacrylate-co-2-amino-ethyl methacrylamide) MALDI-TOF 100-sample array chips for integrated proteomic sample preparation/measurements have been fabricated using a simple atmospheric molding protocol. The enzyme link on the polymeric chip surface has been created by covalently binding ethylene glycol disuccinate bis(sulfo-N-succinimidyl) ester with the amine functionalities of the chip well surface and subsequent reaction of the linker with 1.3 nmol trypsin. The superior performance of the new chips is demonstrated for the enzymatic digestion of individual proteins (500 fmol) of 5-60 kDa size. A mixture of 500 fmol cytochrome C, bovine serum albumin, human hemoglobin, and horse myoglobin was deposited in the trypsin-linked sample well, followed by 15-60 min of on-chip digestion. Subsequent peptide mass fingerprinting using protein-database-searching software identified all four proteins. The combination of hydrophobic pMALDI arrays and novel enzyme-linked chips minimizes sample-handling times and enhances the analytical information collected by offering intact protein mass measurements combined with enzymatic cleavage and the peptide mass fingerprint. Our concept can be readily extended to further high-throughput enzyme activity screening and protein processing.
Ibañez
Alfredo J
AJ
Mass Spectrometry Research Group, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, 07745 Jena, Germany.
Muck
Alexander
A
Halim
Vincentius
V
Svatos
Ales
A
D000818:Animals; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized / Q000378:metabolism; D006801:Humans; D046208:Microchip Analytical Procedures / Q000379:methods*; D011108:Polymers; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011108:Polymers; D011506:Proteins; D014357:Trypsin