Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion.
A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in "real-world" proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.
Kim
Byoung Chan
BC
Institut Pasteur Korea, Seoul, Republic of Korea.
Lopez-Ferrer
Daniel
D
Lee
Sang-Mok
SM
Ahn
Hye-Kyung
HK
Nair
Sujith
S
Kim
Seong H
SH
Kim
Beom Soo
BS
Petritis
Konstantinos
K
Camp
David G
DG
Grate
Jay W
JW
Smith
Richard D
RD
Koo
Yoon-Mo
YM
Gu
Man Bock
MB
Kim
Jungbae
J
D055162:Biocatalysis; D019149:Bioreactors; D002853:Chromatography, Liquid; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D017743:Equipment Reuse; D008855:Microscopy, Electron, Scanning; D049329:Nanostructures / Q000648:ultrastructure; D010446:Peptide Fragments; D011108:Polymers / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism*
D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011108:Polymers; D011506:Proteins; D014357:Trypsin