Jos Jansen

5 113 Screening for abnormal glycosylation in a cohort of adult liver disease patients We defined abnormal TF sialylation as an increased percentage of hyposialylated TF isoforms compared with the main isoform, tetrasialoTF. Control ranges were used from the clinical diagnostic protocol, derived by tIEF of 59 healthy control samples: asialoTF 0-3.2%; monosialoTF 0-5.0%; disialoTF 3.3-7.6%; trisialoTF 4.9- 10.6%; pentasialoTF 18.7-31.5%. If applicable, wedesignated theprofile type1or type2CDGbasedon international consensus. Type 1 CDG has increased asialo- and disialo TF isoforms, indicating loss of 1 or 2 glycans. Type 2 CDG has hyposialylation for all TF isoforms.(16) Abnormal tIEF samples were selected for further work-up with QTOF-MS. Quality criteria for inclusion of the sample included an abundance of > 50.000 amu of the intact TF glycoprotein (79556 amu). We selected peaks corresponding to a known TF isoform with an abundance of > 1000. The relative abundance of these peaks was calculated based on their percentage relative to total abundance. Figure 1 shows a typical tIEF and QTOF-MS pattern and depicts the nomenclature of the glycosylation isoforms used throughout this manuscript. Figure 1. Overview of a normal tIEF and QTOF-MS profile . A. Typical tIEF pattern. The most abundant fraction correlates with the intact TF glycoprotein. B Typical QTOF-MS profile of intact TF with two attached glycans. Shown are the most commonly encountered glycans. The green horizontal bar corresponds with the amino acid backbone. The peak at 79556 amu correlates with the intact TF glycoprotein. The legend for the monosaccharides is: blue square: N-acetylglucosamine, red triangle: fucose, green circle: mannose, yellow circle: galactose, purple diamond: sialic acid. Sample preparation for N-glycan analysis Transferrin isoelectric focusing All samples were analyzed with tIEF to determine abnormal sialylation of TF. tIEF was performed as described before. (14) Briefly, serum or plasma samples were incubated with iron and applied to a 5-7 pH gradient gel for electrophoresis. After completion, gels were incubated with 2.5 µl/cm2 anti-TF antibody (Dako