4.4. Mass Spectrometry (MS) Analysis of Microsamples
Sample preparation was automated on AssayMap BRAVO (Agilent Technologies, Santa Clara, CA, USA) to reduce preanalytical variability. Briefly, proteins from a 6 mm DBS card punch were extracted with ammonium bicarbonate (50 mM) while plasma microsamples (2 µL) were initially treated with denaturing buffer. Protein samples were reduced and alkylated, cleaned according to a proprietary protocol (WO/2020/234287) and digested with trypsin prior to LC-MS analysis, as shown in Dried samples were resuspended in an acetonitrile/formic acid–water mixture (2.0/0.1/97.9%) spiked with reference peptides (The Opensource Skyline