Identification of N-linked glycosylation sites in human nephrin using mass spectrometry.
Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.
Khoshnoodi
Jamshid
J
Department of Medicine, Vanderbilt University Medical School, Nashville, Tennessee 37232-2372, USA. jamshid.khoshnoodi@vanderbilt.edu
Hill
Salisha
S
Tryggvason
Karl
K
Hudson
Billy
B
Friedman
David B
DB
D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002241:Carbohydrates; D008565:Membrane Proteins; C113267:nephrin