Identification and analysis of o-acetylated sialoglycoproteins

Methods Mol Biol. 2013;981:57-93. doi: 10.1007/978-1-62703-305-3_6.

Abstract

5-N-acetylneuraminic acid, commonly known as sialic acid (Sia), constitutes a family of N- and O-substituted 9-carbon monosaccharides. Frequent modification of O-acetylations at positions C-7, C-8, or C-9 of Sias generates a family of O-acetylated sialic acid (O-AcSia) and plays crucial roles in many cellular events like cell-cell adhesion, proliferation, migration, etc. Therefore, identification and analysis of O-acetylated sialoglycoproteins (O-AcSGPs) are important. In this chapter, we describe several approaches for successful identification of O-AcSGPs. We broadly divide them into two categories, i.e., invasive and noninvasive methods. Several O-AcSias-binding probes are used for this purpose. Detailed methodologies for step-by-step identification using these probes have been discussed. We have also included a few invasive analytical methods for identification and quantitation of O-AcSias. Several indirect methods are also elaborated for such purpose, in which O-acetyl group from sialic acids is initially removed followed by detection of Sias by several approaches. For molecular identification, we have described methods for affinity purification of O-AcSGPs using an O-AcSias-binding lectin as an affinity matrix followed by sequencing using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF) mass spectroscopy (MS). In spite of special attention, loss of O-acetyl groups due to its sensitivity towards alkaline pH and high temperature along with migration of labile O-acetyl groups from C7-C8-C9 during sample preparation is difficult to avoid. Therefore there is always a risk for underestimation of O-AcSias.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Animals
  • Chromatography, Affinity / methods*
  • Chromatography, High Pressure Liquid
  • Erythrocytes / metabolism
  • Hemolymph / metabolism
  • Humans
  • Lectins / analysis
  • Lectins / chemistry
  • Lectins / metabolism
  • N-Acetylneuraminic Acid / analysis
  • Sialoglycoproteins / analysis
  • Sialoglycoproteins / chemistry
  • Sialoglycoproteins / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*

Substances

  • Lectins
  • Sialoglycoproteins
  • N-Acetylneuraminic Acid