Ten microliters of plasma sample were placed into a clean Eppendorf tube followed by the addition of 80 μL MeOH to precipitate the protein. The methanol was removed after centrifugation and the pellet was air-dried and afterwards re-suspended in 50 mM NH4HCO3/0.16% ProteaseMAX containing a heavy-isotope labeled peptide, which is specific for human Hpx and is used as internal standard. After incubation at 56°C/550 rpm for 45 min the samples were reduced by adding 0.5 M DTT (56°C/550 rpm for 20 min). The samples were then alkylated by addition of 0.5 M IAA and incubation for 20 min at RT protected from light. Tryptic digestion was carried out at 37°C/550 rpm and stopped after 3 h by addition of formic acid. After centrifugation the samples were separated immediately on a C18 column (AdvanceBio Peptide Mapping, 2.1 × 150 mm). The measurements were conducted using an Agilent 1290 Infinity II – 6550 iFunnel QTOF LC-MS system.
Data was analyzed by calculating the peak area of the analyte and the internal standard using Agilent MassHunter Quant software. A standard curve was created in Agilent MassHunter Quant by plotting the average response ratio of analyte to internal standard against concentration for each standard sample. The analyte concentration in the plasma samples was backcalculated using the standard curve equation.
Data was analyzed by calculating the peak area of the analyte and the internal standard using Agilent MassHunter Quant software. A standard curve was created in Agilent MassHunter Quant by plotting the average response ratio of analyte to internal standard against concentration for each standard sample. The analyte concentration in the plasma samples was backcalculated using the standard curve equation.