LCMS-based Validation of Glycosylation Variants in Transferrin – A Diagnostic Approach for Congenital Disorders of Glycosylation

Abstract

Congenital disorders of glycosylation (CDG) are rare genetic diseases caused by defects in the glycosylation pathways. Early and accurate diagnosis of congenital disorders of glycosylation (CDG) is crucial for timely initiating appropriate therapies, which can significantly improve clinical outcomes. Our research aims to develop a fast and reliable diagnostic test for CDG Carbohydrate-deficient transferrin (CDT) is a key biochemical marker in the diagnosis of CDG. Transferrin (TF) is an iron-transport protein that contains N-glycans at Asn432 and Asn630 in humans. It is important to note that CDT represents a group of transferrin sialoforms, including asialo-, monosialo-, disialo-, and rarely trisialo-TF. In this study, we developed and validated a targeted proteomics assay using liquid chromatography-mass spectrometry (LC/MS/MS) to measure transferrin glycosylation variants in human serum. We selected the tryptic peptides 421CGLVPVLAENYNK433 and 622QQQHLFGSNVTDCSGNFCLFR642 as analytes for quantification, each containing glycosylation variants at N-432 and N-630, respectively. Additionally, we evaluated the feasibility of directly quantifying
peptides for transferrin sialoforms using selected reaction monitoring (SRM) –Mass Spectrometry (MS) analysis