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 Frechet*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

59 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

Keywords

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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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