IRAK4 degradation in blood and skin by MS
dryness
LYSED
Targeted mass spectrometry (LC–PRM/MS) was performed at Biognosys on isolated PBMCs collected from blood samples (6 ml of K2EDTA) or frozen skin biopsies. PBMCs were isolated at the clinical site using the Ficoll-paque filled Leucosep system, and, once purified, the PBMC pellets were frozen and shipped to Biognosys where the PBMC pellet was lysed and denatured using Biognosys Denature Buffer. The 5-mm skin biopsies were collected, trisected from the dermis to the epidermis and immediately frozen. One of the frozen aliquots was shipped to Biognosys where the tissue was homogenized with a Precellys homogenizer, and proteins were denatured using Biognosys Denature Buffer. Protein concentrations for both PBMCs and skin were determined by bicinchoninic acid (BCA) assay and then reduced and alkylated for 60 min at 37 °C, followed by overnight incubation with trypsin. C18 cleanup for MS was carried out using BioPureSPE MINI spin columns. Peptides were evaporated to complete dryness, and peptide concentrations were measured using the micro BCA assay. Before LC–PRM/MS analysis, sample preparations were spiked with the SIS peptide pool at a concentration of 30 fmol µgSignal processing and data analysis were carried out using SpectroDive software. Peak integration, retention time alignment and peak scoring were passed on mProphet