Glycoprotein analysis using enzymatic digestion and MALDI-TOF MS

Rich Kornfeld; James W. Kenny; Scot R. Weinberger; Yi Yang; Ron Orlando

doi:10.1117/12.237629

1 April 1996 Glycoprotein analysis using enzymatic digestion and MALDI-TOF MS

Rich Kornfeld, James W. Kenny, Scot R. Weinberger, Yi Yang, Ron Orlando

Proceedings Volume 2680, Ultrasensitive Biochemical Diagnostics; (1996) https://doi.org/10.1117/12.237629
Event: Photonics West '96, 1996, San Jose, CA, United States

Abstract

A sensitive and facile method is described to identify the glycosylation sites and site-specific heterogeneity in the carbohydrate portion of glycoproteins. In this procedure, the peptide backbone of the glycoprotein is cleaved enzymatically. The peptide/glycopeptide mixture is divided into three fractions. The first fraction is analyzed directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), while the other two aliquots are analyzed by MALDI-TOF MS after enzymatic release of the N-linked and N- and O-linked chains. Comparison of these mass spectra provides the molecular weight of each carbohydrate side chain and of the peptide to which it is attached. This information combined with the protein's amino acid sequence identifies the glycosylation sites and provides information concerning site-specific oligosaccharide heterogeneity. This approach is faster and simpler than procedures currently used for glycosylation site mapping and can be performed on as little as 10 picomoles of glycoprotein.

Citation Download Citation

Rich Kornfeld, James W. Kenny, Scot R. Weinberger, Yi Yang, and Ron Orlando "Glycoprotein analysis using enzymatic digestion and MALDI-TOF MS", Proc. SPIE 2680, Ultrasensitive Biochemical Diagnostics, (1 April 1996); https://doi.org/10.1117/12.237629

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