Rapid Determination of Transferrin Isoforms by Immunoaffinity Liquid Chromatography and Electrospray Mass Spectrometry

Abstract

Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography–mass spectrometry (LC-MS). Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. Results: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and interassay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018–0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002–0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170). Conclusions: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.

Identification of a frequent variant inALG6, the cause of Congenital Disorder of Glycosylation-Ic

“…What is unclear is whether this variation actually causes CDG-Ic. The patient described here showed normal Tf glycosylation pattern by IEF and mass spectrometric analysis (Lacey et al, 2001). These results were somewhat surprising, but not unprecedented.…”

Mass spectrometry for protein sialoglycosylation

“…In LC-MS analysis, reversed-phase chromatography is commonly used for separating the subunits or domains of proteins, [91][92][93] and immunoaffinity chromatography for enriching the target compounds. 66 As the glycoproteins with high carbohydrate content present relatively weak binding to hydrophobic stationary phase (reversed-phase chromatography) and sialic acid moieties further enhance the nature, the samples are often eluted at low organic concentrations. Analysis of eluted fractions is performed in positiveion mode with typical m/z at 1000-5000.…”

IGF system in children with congenital disorders of glycosylation

“…Analysis of transferrin glycosylation by immunoisoelectric focusing and more recently by tandem mass spectroscopy has been used as a diagnostic test for CDG. 19,20 Postnatal growth failure is common in children with CDG despite normal or elevated levels of GH. 1,21 In a prospective study of growth in children with CDG-Ia, Kjaergaard et al .…”