Jos Jansen

1 Introduction to hepatic congenital glycosylation disorders 17 protein. Additionally, a lack of total glycans cannot be measured, rendering it less useful in the diagnostics for type 1 CDG. Additionally, because of the labile nature of the sialic acid, the glycan has to be derivatized to improve its stability during MS. This is less of a problem in recent LC-MS glycomics methods. QTOF mass spectrometry analysis of intact transferrin Efforts to overcome the limitations of IEF and whole serumN-glycan analysis lead to implementation and validation of mass spectrometry of the serum transferrin glycoprotein.(57) In short, serum transferrin is pulled down with anti-transferrin beads and injected on a LC system, before being sprayed into the Quadruple Time of Flight mass spectrometer (QTOF MS). A typical QTOF-MS spectrum of the intact transferrin is shown in Figure 3. Any peak with lower molecular mass represent incomplete glycans. The difference on the x-axis correlates to the weight of specific sugars. For example: loss of one sialic acid (296 amu), typical for type 2 CDG. Reference ranges of the percentage of the main peak have been determined for diagnostic purposes. The advantage of mass spectrometric methods has been shown by the ability to unravel the pathogenic defect in several CDG subtypes. Two examples are MAN1B1-CDG and PGM1-CDG. MAN1B1 is the enzyme that cleaves offmannoses during glycanmodification.(58) QTOFMS profiling showed accumulation of peaks associated with uncleaved mannose glycans. Another example is PGM1-CDG. (59) PGM1 catalyzes the bi-directional interconversion of glucose-1-phosphate and glucose-6-phosphate and is dually important for glucose metabolism and glycosylation. QTOF-MS showed a combination of type 1 and type 2 CDG, possibly due to effects on several events in the glycosylation pathway. Additionally, oral galactose supplementation in PGM1-CDG could be monitored by QTOF glycoprofiling. QTOF-MS can also be valuable in identification and grouping of CDG with an unknown genetic cause. Disadvantages are that only the glycosylation of transferrin can be measured. This implies that tissue- or protein-specific glycosylation defects, such as the Cohen syndrome, are missed. (60) This limitation extends to cell models as only hepatic cell lines produce transferrin and glycosylation of transferrin does not resemble physiological transferrin (unpublished observations).