Trypsin coatings on electrospun and alcohol-dispersed polymer nanofibers for trypsin digestion column
The construction of a trypsin reactor in a chromatography column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coating, which was prepared by a two-step process of covalent attachment and enzyme crosslinking. In a comparative study with the trypsin coatings on asspun and non-dispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than twenty minutes. By applying the alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the enzymes were improved yielding more complete and reproducible digestions. Regardless of alcohol-dispersion or not, trypsin coating showed better digestion performance and improved performance stability under recycled uses than covalently-attached trypsin. The combination of highly stable trypsin coating and alcoholdispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for on-line and automated protein digestion.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 990123
- Report Number(s):
- PNNL-SA-72383; 32707; KP1601010; TRN: US201020%%133
- Journal Information:
- Analytical Chemistry, 82(18):7828 - 7834, Vol. 82, Issue 18
- Country of Publication:
- United States
- Language:
- English
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