期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1188, 期 2, 页码 88-96出版社
ELSEVIER
DOI: 10.1016/j.chroma.2008.02.075
关键词
nanoHPLC; enzymatic reactor; monolithic columns; mass spectrometry; pepsin; immobilized enzyme
资金
- NIGMS NIH HHS [GM44885, R01 GM044885-15, R01 GM044885, R01 GM048364-16] Funding Source: Medline
The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate-co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions. (C) 2008 Published by Elsevier B.V.
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