A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides.
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.
Research Institute of the Hospital for Sick Children, Toronto, Ont., Canada M5G 1X8.
Xiaolian
X
Fan
Yi-Min
YM
She
Rick D
RD
Bagshaw
John W
JW
Callahan
Harry
H
Schachter
Don J
DJ
Mahuran
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029742:Caenorhabditis elegans Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D001216:Asparagine