Capillary column chromatography improves sample preparation for mass spectrometric analysis: complete characterization of human alpha-enolase from two-dimensional gels following in situ proteolytic digestion

Electrophoresis
G E ReidR J Simpson

Abstract

Two-dimensional polyacrylamide gel electrophoresis (2-DE) in combination with mass spectrometry is an extremely powerful tool for characterizing complex mixtures of proteins. In many cases, the success of this approach relies upon the ability to recover peptides at high concentrations and free of interfering artifacts from in-gel and/or on-membrane enzymatic digests. In previous studies, we demonstrated that capillary or microcolumn (< 350 microm ID) reversed-phase high performance liquid chromatography (RP-HPLC) is a powerful microseparation technique for proteins and peptides (Moritz, R. L. and Simpson, R. J., J. Chromatogr. 1992, 599, 119-130). Here we evaluate various capillary column RP-HPLC/mass spectrometric approaches for identifying and characterizing 2-DE resolved proteins. For these studies, stable and efficient 0.20 mm and 0.32 mm internal diameter (ID) fused-silica columns with hydrophilic polyvinylidene difluoride (PVDF) frits were fabricated and slurry packed with 7 microm spherical, 300 A pore size, C8 bonded phase silica particles. We show that capillary column chromatography is a rapid and efficient desalting/concentrating (ON/OFF) technique for sample cleanup prior to protein identification by peptide-mass fi...Continue Reading

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Citations

Mar 10, 2001·Rapid Communications in Mass Spectrometry : RCM·J Delaney, P Vouros
Dec 16, 2000·Journal of the American Society for Mass Spectrometry·G E ReidR A O'Hair
Jul 9, 2003·Proceedings of the National Academy of Sciences of the United States of America·Harshal H NandurkarChristina A Mitchell
Nov 21, 2012·PloS One·Ignazio Garaguso, Juergen Borlak

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