53 LC-MS/MS-method development quantifying FVIII Instrumentation and chromatographic conditions Sample purification was performed on a vibramax 100 plate shaker (Heidolph Instruments, Schwabach, Germany). Sample digestion was performed on a ThermoMixer (Eppendorf, Nijmegen, the Netherlands). All experiments were performed on an Vanquish UHPLC coupled to a TSQ Altis (Thermo Fisher, Waltham, MA, USA). The analytical column was AcclaimTM, RSLC 120, C18, 2.1 x 100 mm, 2.2 μm particle size obtained from Thermo Fisher and was maintained at 50 °C. The mobile phases were: (a) 0.1 % formic acid in water; (b) 0.1 % formic acid in ACN. The LC gradients in minutes per percentage of mobile phase B were 0.0 (min)/10 (% B), 8/25, 8.1/80, 10/80, 10.1/10 and 12/10. The flow rate was 0.6 mL/min and the run time was 9 min. The MS was operated in positive mode with spray voltage of 2.5 kV, Ion Transfer Tube Temperature 400 °C, vaporizer temperature 350 °C, aux gas pressure 20 Arb, sheath gas pressure 40 Arb, sweep gas pressure 0 Arb and collision gas pressure 2.5 mTorr. The precursor ions, product ions, collision energy and radio frequency (RF) lens settings are listed in Table 1 for FVIII signature peptide and for the stable isotopic labeled internal standard. Table 1. TSQ Altis Mass Spectrometry Conditions for SRM transitions for the signature peptide liberated from FVIII after digestion with trypsin and the internal standard stable isotopic labelled FVIII peptide. Peptide sequence Precursor charge Precursor (m/z) Product charge Product (m/z) Product- Ion type CE (V) RF (V) Dwell time (ms) GELNEHLGLLGPYIR 3+ 560.97 2+ 747.93 Y5 16 65 340 GELNEHLGLLGPYIR[13C6,15N4], (IS) 3+ 564.31 2+ 752.93 Y5 16 65 60 Notes: both sequences had a retention time of 7.2 min. Abbreviations: CE: collision energy; RF: radio frequency lens; IS: internal standard. Sample preparation Sample preparation was based on immunoaffinity purification where the light chain of FVIIIa was captured by means of a biotinylated camelid nanobody (b-anti-FVIII) which in turn was bound to a streptavidin coated 96 well plate (Figure 1). In day 1, b-anti-FVIII was coupled to a streptavidin coated 96 well plate by pipetting 200 µL b-anti-FVIII (1 ng/µL) dissolved in PBS, 0.05% Tween-20, 0.1% BSA in each well, followed by 3 hour binding on a plate shaker (300 rpm) at room temperature. The plate was washed 3 times with 200 µL PBS (0.1% Tween-20, 0.1% BSA) and stored upside down in a zip lock bag at -80 °C. On day 2, 100 uL(micro symbool) PBS (0.05% Tween-20, 0.1% BSA) and 50 uL(ook microsymbool hier) sample (standard or QC) were pipetted to the antibody coated 96 well plate. Then, VWF was dissociated from FVIII by adding 2.5 uL(ook weer micro symbool) (Ca 2.5 M + thrombin 500 IE/mL) followed by an overnight incubation at 400 rpm on a plate shaker. The next day, the wells were washed three times with 200 µL PBS (0.05% Tween-20, 0.1% BSA). Then, 100 µL IS solution (5 ng/mL) in elution solvent (0.1% FA with 0.005% zwittergent 3-16 dissolved in water) was added to each well and mixed for 5 min at 3
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