Reduction-Free Thermolysin Digestion Enables Absolute Protein Quantitation in Plasma in 35 min from Sample to Result

We previously reported that the broad-specificity protease thermolysin yields reproducible, near-complete proteome digests within 1-2 min. Here, we demonstrate rapid absolute protein quantitation in human plasma by combining reduction/alkylation-free thermolysin digestion on S-Trap cartridges with full-length stable isotope-labeled (SIL) protein standards and parallel reaction monitoring (PRM). Post-digest dilution of S-Trap eluates enabled immediate EvoTip loading, yielding LC-MS-ready samples in under 20 min. Using the anti-CD20 monoclonal antibody rituximab as a showcase, a lower limit of quantitation of 5 amol on-column per ∼250 ng plasma protein injection could be achieved on an Orbitrap Exploris 480 in 60/100 samples-per-day mode. We applied the assay to 23 total pretreatment and on-treatment plasma samples from 12 chronic lymphocytic leukemia patients, demonstrating fit-for-purpose absolute quantitation in a clinical matrix. In 100 samples-per-day mode, the full workflow enables on-demand absolute protein quantitation in 35 min from sample to fully acquired LC-MS data. The achieved sensitivity indicates the feasibility of transfer to other targets or matrices with lower concentrations. Because the required SIL proteins are used at femtomole levels (here, 40 fmol of SIL rituximab spiked into 10 μg of plasma protein), a single 10-μg SIL IgG vial is sufficient as internal standard for ∼1700 injections.