PMID- 28213235
OWN - NLM
STAT- MEDLINE
DCOM- 20180905
LR  - 20180905
IS  - 1873-3573 (Electronic)
IS  - 0039-9140 (Linking)
VI  - 166
DP  - 2017 May 1
TI  - Preparation and characterization of a packed bead immobilized trypsin reactor 
      integrated into a PDMS microfluidic chip for rapid protein digestion.
PG  - 275-283
LID - S0039-9140(17)30172-8 [pii]
LID - 10.1016/j.talanta.2017.01.060 [doi]
AB  - This paper demonstrates the design, efficiency and applicability of a simple, 
      inexpensive and high sample throughput microchip immobilized enzymatic reactor 
      (IMER) for rapid protein digestion. The IMER contains conventional silica 
      particles with covalently immobilized trypsin packed inside of a 
      poly(dimethylsiloxane) (PDMS) microchip channel (10mmx1mmx35microm). The microchip 
      consists of 9 different channels, enabling 9 simultaneous protein digestions. 
      Trypsin was covalently immobilized using carbodiimide activation, the ideal 
      trypsin/silica particle ratio (i.e. measured mass ratio before the immobilization 
      reaction) was determined. The amount of immobilized trypsin was 10-15mug trypsin 
      for 1mg silica particle. Migration times of CZE peptide maps showed good 
      repeatability and reproducibility (RSD%=0.02-0.31%). The IMER maintained its 
      activity for 2 months, in this period it was used effectively for rapid 
      proteolysis. Four proteins (myoglobin, lysozyme, hemoglobin and albumin) in a 
      wide size range (15-70kDa) were digested to demonstrate the applicability of the 
      reactor. Their CZE peptide maps were compared to peptide maps obtained from 
      standard in-solution digestion of the four proteins. The number of peptide peaks 
      correlated well with the theoretically expected peptide number in both cases, the 
      peak patterns of the electropherograms were similar, however, digestion with the 
      microchip IMER requires only <10s, while in-solution digestion takes 16h. 
      LC-MS/MS peptide mapping was also carried out, the four proteins were identified 
      with satisfying sequence coverages (29-50%), trypsin autolysis peptides were not 
      detected. The protein content of human serum was digested with the IMER and with 
      in-solution digestion.
CI  - Copyright A(c) 2017 Elsevier B.V. All rights reserved.
FAU - Kecskemeti, Adam
AU  - Kecskemeti A
AD  - Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem 
      ter 1, Debrecen 4032, Hungary.
FAU - Gaspar, Attila
AU  - Gaspar A
AD  - Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem 
      ter 1, Debrecen 4032, Hungary. Electronic address: gaspar@science.unideb.hu.
LA  - eng
PT  - Journal Article
DEP - 20170125
PL  - Netherlands
TA  - Talanta
JT  - Talanta
JID - 2984816R
RN  - 0 (Dimethylpolysiloxanes)
RN  - 0 (Enzymes, Immobilized)
RN  - 0 (Peptides)
RN  - 63148-62-9 (baysilon)
RN  - EC 3.4.21.4 (Trypsin)
SB  - IM
MH  - Dimethylpolysiloxanes/*chemistry
MH  - Enzymes, Immobilized/*chemistry/*metabolism
MH  - *Lab-On-A-Chip Devices
MH  - Microspheres
MH  - Peptides/metabolism
MH  - *Proteolysis
MH  - Time Factors
MH  - Trypsin/*chemistry/*metabolism
OTO - NOTNLM
OT  - Microchip reactor
OT  - Peptide mapping
OT  - Protein digestion
OT  - Trypsin immobilization
EDAT- 2017/02/19 06:00
MHDA- 2018/09/06 06:00
CRDT- 2017/02/19 06:00
PHST- 2016/11/25 00:00 [received]
PHST- 2017/01/20 00:00 [revised]
PHST- 2017/01/24 00:00 [accepted]
PHST- 2017/02/19 06:00 [entrez]
PHST- 2017/02/19 06:00 [pubmed]
PHST- 2018/09/06 06:00 [medline]
AID - S0039-9140(17)30172-8 [pii]
AID - 10.1016/j.talanta.2017.01.060 [doi]
PST - ppublish
SO  - Talanta. 2017 May 1;166:275-283. doi: 10.1016/j.talanta.2017.01.060. Epub 2017 
      Jan 25.