In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy

Laurent Geiser, Sebastiaan Eeltink, Frantisek Svec, Jean M.J. Fréchet*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)88-96
Number of pages9
JournalJournal of Chromatography A
Volume1188
Issue number2
DOIs
StatePublished - Apr 25 2008
Externally publishedYes

Bibliographical note

Funding Information:
Support by grants of the National Institute of General Medical Sciences, National Institutes of Health (GM44885) and Pfizer Inc. is gratefully acknowledged. Characterization work at the Molecular Foundry was supported by the Director, Office of Science, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, of the US Department of Energy under contract No. DE-AC02-05CH11231. Insightful discussions with Violaine Augustin and Timothy Stachowiak are gratefully acknowledged.

Keywords

  • Enzymatic reactor
  • Immobilized enzyme
  • Mass spectrometry
  • Monolithic columns
  • NanoHPLC
  • Pepsin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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