Rapid determination of transferrin isoforms by immunoaffinity liquid chromatography and electrospray mass spectrometry.
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 microL) was diluted with 100 microL 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.
Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology, Biomedical Mass Spectrometry and Functional Proteomics Facility, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA.
J M
JM
Lacey
H R
HR
Bergen
M J
MJ
Magera
S
S
Naylor
J F
JF
O'Brien
D000818:Animals; D000906:Antibodies; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing; D020033:Protein Isoforms / Q000097:blood / Q000276:immunology; D011817:Rabbits; D012016:Reference Values; D021241:Spectrometry, Mass, Electrospray Ionization; D014168:Transferrin / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D020033:Protein Isoforms; D014168:Transferrin