Efficient protein digestion with peptide separation in a micro-device interfaced to electrospray mass spectrometry

Haider Al-Lawati, Paul Watts, Kevin J. Welham

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

A highly efficient protein digestion device has been fabricated using commercially available immobilized trypsin on agarose beads, packed into a silica capillary and connected either directly to an electrospray mass spectrometer via a 'microtight T' connector, from which aqueous acetic acid (0.2%) was pumped, or via a monolithic column connected to the mass spectrometer ion source. Six proteins with molecular mass ranging from 2848 to 77703 Da were digested completely using this system. In the second set of experiments a short monolithic separation column was placed after the immobilized trypsin capillary and partial separation of the generated peptides was obtained. The detection limits were increased from the μmol to pmol range by utilization of this separation column. Gradient elution, using a binary HPLC pump and a flow splitter, was used to optimize the peptide separation. This provided significantly enhanced resolution of the tryptic peptides but increased the analysis time to 30 minutes.

Original languageEnglish
Pages (from-to)656-663
Number of pages8
JournalAnalyst
Volume131
Issue number5
DOIs
Publication statusPublished - 2006

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ASJC Scopus subject areas

  • Analytical Chemistry

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