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001-es BibID:BIBFORM068560
Első szerző:Kecskeméti Ádám (vegyész)
Cím:Preparation and characterization of a packed bead immobilized trypsin reactor integrated into a PDMS microfluidic chip for rapid protein digestion / Ádám Kecskeméti, Attila Gáspár
Megjegyzések:This paper demonstrates the design, efficiency and applicability of a simple, inexpensive and high samplethroughput microchip immobilized enzymatic reactor (IMER) for rapid protein digestion. The IMER containsconventional silica particles with covalently immobilized trypsin packed inside of a poly(dimethylsiloxane)(PDMS) microchip channel (10 mm?1 mm?35 ?m). The microchip consists of 9 different channels, enabling 9simultaneous protein digestions. Trypsin was covalently immobilized using carbodiimide activation, the idealtrypsin/silica particle ratio (i.e. measured mass ratio before the immobilization reaction) was determined. Theamount of immobilized trypsin was 10?15 ?g trypsin for 1 mg silica particle. Migration times of CZE peptidemaps showed good repeatability and reproducibility (RSD%=0.02?0.31%). The IMER maintained its activity for2 months, in this period it was used effectively for rapid proteolysis. Four proteins (myoglobin, lysozyme,hemoglobin and albumin) in a wide size range (15?70 kDa) were digested to demonstrate the applicability ofthe reactor. Their CZE peptide maps were compared to peptide maps obtained from standard in-solutiondigestion of the four proteins. The number of peptide peaks correlated well with the theoretically expectedpeptide number in both cases, the peak patterns of the electropherograms were similar, however, digestion withthe microchip IMER requires only < 10 s, while in-solution digestion takes 16 h. LC-MS/MS peptide mappingwas also carried out, the four proteins were identified with satisfying sequence coverages (29?50%), trypsinautolysis peptides were not detected. The protein content of human serum was digested with the IMER and within-solution digestion.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
Trypsin immobilization
Protein digestion
Peptide mapping
Microchip reactor
Megjelenés:Talanta. - 166 (2017), p. 275-283. -
További szerzők:Gáspár Attila (1970-) (vegyész, kémikus)
Pályázati támogatás:OTKA-111932
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