Jos Jansen

114 Chapter 5 #A0061, Carpinteria, CA USA) and visualized with Coomassie blue. Data analysis was performed with Image Quant Software (Totallab, Newcastle upon Tyne, UK) TF polymorphisms were recognized by doubled bands for all isoforms. For confirmation, neuraminidase treatment was applied on samples with ambiguous isoelectric patterns. (14) Nano liquid chromatography-chip (C8)- quadruple time of flight mass spectrometry (QTOF-MS) Mass spectrometry analysis was performed as described before.(15) First, beads were loadedwith anti-TF antibody (Dako #A0061, Carpinteria, CAUSA) and stored in 20% ethanol. Prior to usage, beads were washed 4 times with a Tris-HCl (pH 7) solution. Next, 100 µl of a 1:10 plasma sample dilution in 0.9% sodiumchloride was mixed with beads and incubated for 20 minutes under continuous shaking at 3000 rpm/min. Subsequently beads were washed four times with Tris-HCl (pH 7) solution. For elution, 1 µl of Tris-HCl pH 9 solution was added to the sample followed by 50 µl elution buffer (0.1M Glycine-HCl pH 2.7). After spinning and verification of neutral pH, 2µl sample was injected into the microfluidic 6540 HPLC-chip-QTOF instrument (Agilent Technologies, Santa Clara, CA, USA). For data analysis of QTOF-MS profiles Agilent Mass Hunter Qualitative Analysis software (v. B.04.00) was used. Statistical analysis For statistical analysis SPSS Statistics v. 22 (IBM Corporation, Armonk, NY, USA) was used. Due to non-linearity of our data we used the non-parametric Kruskal- Wallis test for comparisons of more than two groups and the Mann-Whitney U test when there were two groups. P-values were adjusted for multiple testing with the Bonferroni method.