Pharmaceutical Sciences Faculty Publications

Stepwise Mobilization of Focused Proteins in Capillary Isoelectric Focusing Mass Spectrometry

Document Type

Article

Publication Date

4-1-2000

Journal Title

Analytical Chemistry

ISSN

0003-2700

Volume

72

Issue

7

First Page

1462

Last Page

1468

DOI

10.1021/ac9912653

PubMed ID

10763241

Abstract

A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.

Keywords

Electrophoresis, capillary, isoelectric focusing, mass spectrometry, proteins

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