A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides

Anal Biochem. 2004 Sep 1;332(1):178-86. doi: 10.1016/j.ab.2004.05.038.

Abstract

Glycosylated proteins on the cell surface have been shown to be essential for cell-cell interactions in development and differentiation. Our ultimate goal is to identify Asn-linked oligosaccharides that are directly involved in these critical in vivo functions. Because such oligosaccharides would be expected to reside on the integral plasma membrane proteins, and conventional two-dimensional gel techniques are ineffective at separating such proteins, we have developed a new approach to their identification on a proteomics scale from Caenorhabditis elegans. Membrane proteins are solubilized in guanidine-HCl, precipitated, and digested with trypsin. The glycopeptides are then separated by lectin chromatography. Next, glycopeptidase F digestion removes the oligosaccharides from the peptides and converts to Asp each Asn to which one was attached. The peptides are then analyzed by matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) mass spectrometry. Thus, the membrane glycoproteins are identified through the sequence tags of these peptides and the conversion of at least one deduced Asn residue to Asp at the Asn-X-Ser/Thr consensus sequence. To validate the utility of this approach, we have identified 13 membrane-bound N-glycosylated proteins from the major peaks observed on MALDI-Q-TOF analysis of our total glycopeptide fraction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Asparagine / metabolism*
  • Caenorhabditis elegans / metabolism
  • Caenorhabditis elegans Proteins / metabolism
  • Glycoproteins / isolation & purification
  • Glycoproteins / metabolism
  • Membrane Proteins / isolation & purification*
  • Membrane Proteins / metabolism
  • Oligosaccharides / metabolism*
  • Peptide Fragments / isolation & purification
  • Peptide Fragments / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Caenorhabditis elegans Proteins
  • Glycoproteins
  • Membrane Proteins
  • Oligosaccharides
  • Peptide Fragments
  • Asparagine