A New Method for the Rapid Diagnosis of Protein N‑linked Congenital Disorders of Glycosylation

The
Congenital Disorders of Glycosylation (CDG) are a devastating group
of genetic disorders that encompass a spectrum of glycosylation defects
and are characterized by the underglycosylation of or the presence
of abnormal glycans on glycoproteins. The N-linked CDG disorders (Type
I and II) are usually diagnosed in chemical pathology laboratories
by an abnormal serum transferrin isoelectric focusing (IEF) pattern.
Transferrin has been the protein of choice for CDG analysis because
it is well characterized, highly abundant, and easily detected in
plasma. However, IEF provides limited information on the glycosylation
defect and requires a separate and extensive glycan analysis to diagnose
CDG Type II. We have therefore developed a simple bead-based immunoaffinity
and mass spectrometry-based assay to address these issues. Our method
uses immuno-purified transferrin and proteolytic digestion followed
by a rapid 30 min mass spectral analysis and allows us to identify
both micro- and macroheterogeneity of transferrin by sequencing of
peptides and glycopeptides. In summary, we have developed a simple,
rapid test for N-linked glycosylation disorders that is a significant
improvement on existing laboratory tests currently used for investigating
defective N-linked glycosylation.