Gradient chromatofocusing-mass spectrometry: a new technique in protein analysis

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Direct interfacing of ion-exchange chromatography with mass spectrometry for protein analysis is rarely reported.
• Gradient elution schemes directly interfaced to mass spectrometry for protein separation are novel.

Purpose of the Study:

• To develop and demonstrate a new analytical technique, gradient chromatofocusing-mass spectrometry (gCF-MS), for protein determination.
• To establish a direct HPLC-MS interface for ion-exchange chromatography of proteins.
• To validate the technique's accuracy and sensitivity for protein molecular mass determination.

Main Methods:

• Development of gradient chromatofocusing-mass spectrometry (gCF-MS) using ion-exchange HPLC directly interfaced with an electrospray-quadrupole mass spectrometer.
• Utilized a descending linear pH gradient (pH 9 to 2.6) on a DEAE HPLC column with volatile buffer components.
• Employed a post-column signal enhancement solution (8% formic acid in acetonitrile) mixed 1:1 with effluent before mass spectrometry analysis.

Main Results:

• Successfully separated six different proteins: carbonic anhydrase II, enolase, beta-lactoglobulin A, lactoglobulin B, soybean trypsin inhibitor, and amyloglucosidase.
• Achieved highly accurate molecular mass determination for proteins within +/-0.010% to 0.033% (+/-100 to 330 ppm).
• Established peak height total ion current detection limits ranging from 4 to 78 pmol (S/N = 3).

Conclusions:

• The developed gCF-MS technique provides a sensitive and accurate method for protein molecular mass determination.
• The direct HPLC-MS interface for ion-exchange chromatography represents a significant advancement in protein analysis.
• This technique is applicable to the analysis of proteins and other charged molecules, offering broad utility in biochemical research.