The advancements in technology have significantly enhanced proteomics by improving the quality, depth, and timeliness of proteomic data, making mass spectrometry a promising tool for personalized medicine. To enable successful longitudinal population-level monitoring, it is essential to have cost-efficient and convenient sample collection methods that yield reliable results. Microsampling performed by patients in their own homes fulfills this requirement. In this study, we evaluate a novel microsampling device that readily prepares a dried plasma sample from blood collected through a finger prick. The device was assessed quantitatively through a targeted proteomics approach, using a selection of stable isotope-labeled protein fragments as standards for plasma proteins spanning a range from 1,300 µM to 30 nM. All samples were analyzed using selected reaction monitoring to ensure quantitative robustness. The device was assessed both from a storage perspective and across a group of healthy donors to ensure reliable performance between individuals. The dried plasma obtained from the device shows an excellent quantitative correlation with conventional plasma (R > 0.99) and high quantitative precision, with a coefficient of variance (CV) below 10% for 80% of all peptides quantified in the group of healthy donors. All the targets that perform well in the microsampling device also show good long-term stability when stored at room temperature for up to 232 days, further showcasing the potential benefits of collecting samples in a dry format.

All samples from the nine donors and stability experiment were analyzed by loading approximately ten micrograms of peptides onto a Thermo Vanquish NEO (Thermo Fisher Scientific) LC system coupled to a Thermo TSQ Altis (Thermo Fisher Scientific). The LC system was operated in a microflow mode and equipped with a 5 mm trapping column (YMC-Triart C18, P/N: TA12S03-E5H0AU, YMC CO., LTD., Kyoto, Japan), a 150 mm analytical column (YMC-Triart C18, P/N: TA12SP9-15H0AU, YMC CO., LTD.) and a stainless-steel spray needle (PepSep emitter, P/N: PSFSELJ20, Bruker). The mobile phase of the LC system consisted of solvent A (0.1% formic acid (FA)) and solvent B (acetonitrile, 0.1% FA).

Dried samples were allowed to equilibrate at RT before the sample discs were loosened from the microsampling device using tweezers. The discs were placed in a 2.0 ml microtiter plate and incubated in elution buffer (50 mM AmBic, 10 mM TCEP, 1% SDC) for 1 hour in a ThermoMixer at 60°C, 850 rpm. Meanwhile, plasma was diluted 40 times in the elution buffer. Forty microliters of diluted plasma or dried sample eluate was spiked with a pre-titred mixture of qRePs (Supplementary Table 1) and incubated for 30 min at 60°C, 850 rpm in a ThermoMixer. CAA was added to a final conc. of 40 mM, and the samples were incubated in darkness for 30 minutes at RT. Trypsin was added to the samples (1 µg), and the samples were incubated in a ThermoMixer at 37°C, 850 rpm overnight. The digestion was quenched by the addition of FA to a final concentration of 1%. The samples were desalted using a positive pressure system (Biotage® Extrahera™ LV200, Uppsala, Sweden) and the iST-PSI kit (PreOmics, Planegg, Germany) according to manufacturer instructions.