Two-Dimensional Microcolumn HPLC Coupled to a Single-Quadrupole Mass Spectrometer for the Elucidation of Sequence Tags and Peptide Mapping


Journal Article

The system described here uses comprehensive two-dimensional liquid chromatography to separate the mixture of peptides resulting from the enzymatic digestion of a protein. This system couples size exclusion liquid chromatography (SEC) to reversed-phase liquid chromatography (RPLC) to affect first a separation based on size followed by a separation based on hydrophobicity. The microcolumn RPLC allows the use of second-dimension flow rates 20-fold less than previously reported, which concomitantly increases the concentration of the peaks. This second dimension of chromatographic separation is directly coupled to an electrospray mass spectrometer for the detection of peptides as well as for the determination of their molecular weights. High-resolution peptide maps have been from as little as 15 pmol of digested protein. The high sensitivity which results from the increased peak concentrations on the reversed-phase microcolumns permits individual peptides to be sequenced using in-sourse collision-induced dissociation (CID) on this single-quadropole mass spectrometer. A low orifice voltage in the electrospray source's interface region is used for intact peptide molecular weight determination. A high orifice voltage on a subsequent scan dissociates the peptide and determines the sequence of about three residues. Knowing enzyme specificity, peptide molecular weight, and a partial sequence allows protein databases to be searched for the protein's identity with a high level of accuracy. Tryptic digests of bovine serum albumin are used to demonstrate the elucidation of sequence tags as well as for traditional peptide mapping experiments. © 1998 John Wiley & Sons, Inc.

Full Text

Duke Authors

Cited Authors

  • Opiteck, GJ; Jorgenson, JW; Moseley, MA; Anderegg, RJ

Published Date

  • January 1, 1998

Published In

Volume / Issue

  • 10 / 4

Start / End Page

  • 365 - 375

International Standard Serial Number (ISSN)

  • 1040-7685

Digital Object Identifier (DOI)

  • 10.1002/(SICI)1520-667X(1998)10:4<365::AID-MCS6>3.0.CO;2-E

Citation Source

  • Scopus