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Capillary isoelectric focusing–electrospray ionization time-of-flight mass spectrometry for protein analysis

✍ Scribed by Jing Wei; Cheng S. Lee; Iulia M. Lazar; Milton L. Lee

Publisher
John Wiley and Sons
Year
1999
Tongue
English
Weight
88 KB
Volume
11
Category
Article
ISSN
1040-7685

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✦ Synopsis

On-line capillary isoelectric focusing CIEF ᎐electrospray ionization Ž . time-of-flight mass spectrometry ESI᎐TOFMS as a two-dimensional separation Ž system is employed for high-resolution analysis of model proteins myoglobin and . ␤-lactoglobulin and human hemoglobin variants C, S, F, and A. The focused proteins in a polyacrylamide-coated capillary are mobilized by replacing the sodium hydroxide catholyte with a sheath liquid solution containing methanol᎐water᎐acetic acid in a volume ratio of 80:19:1. The use of sheath liquid also establishes the electrical connection at the CIEF capillary terminus, which serves to define the electric field along the CIEF capillary and apply an electric voltage for electrospray ionization. At the end of the CIEF capillary, the mobilized protein zones are analyzed and directly identified by ESI᎐TOFMS. The TOFMS accumulates spectra at 5000 Hz frequency and one averaged spectrum is acquired per second during the CIEF᎐ESI᎐TOFMS measurements.

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