SysQuan Enables Affordable and Comprehensive Tissue-Specific Absolute Quantification of the Human Proteome

Despite major technological advances, quantitative proteomics remains hindered by matrix and batch effects, complex workflows, and lengthy chromatographic separations, limiting detection of subtle but biologically important changes. Cross-laboratory reproducibility is poor, and the high cost and limited availability of stable isotope-labeled (SIL) standards have prevented widespread adoption of absolute quantification.
SysQuan addresses these barriers by leveraging SIL mice as a universal internal standard for scalable absolute quantification of the human proteome. By enabling absolute quantification of ~60% of the human proteome at <0.5 cents per isotopically labeled peptide, SysQuan lowers economic and logistical barriers while improving
robustness. C57BL/6 mice were fed a 13C-lysine–enriched diet to generate SIL reference material. Human plasma and tissues, as well as SIL mouse plasma and tissues, were lysed in SDS buffer and mixed 1:1 based on protein concentration. Disulfide bonds were reduced with TCEP, cysteines alkylated with iodoacetamide, and
proteins digested overnight with trypsin using S-Trap columns. Targeted analyses were performed by dynamic MRM on an Agilent 6495D triple quadrupole coupled to an Evosep One LC system. Untargeted analyses employed a timsTOF HT and an Orbitrap Exploris 480, both interfaced with an Evosep One. Mixed human/SIL mouse plasma and tissue digests analyzed by 2D-LC-MS/MS yielded >1,470 human plasma proteins quantifiable from >5,000 co-detected SIL peptide counterparts. In kidney, liver, and lung, 9,830, 7,680, and 9,620 proteins were identified with >66,800, >51,400, and >61,900 paired light/SIL peptides, respectively. Using synthetic light peptides, we developed >2,650 MRM assays for kidney and liver, and >2,580 and 1,786 assays for lung and plasma. Leveraging light peptide standards, we are performing reverse quantification of thousands of SIL mouse proteins with validation following CPTAC guidelines. Validated protein quantities in lyophilized SIL mouse plasma enable absolute quantification of the human plasma proteome in single-acquisition workflows, increasing throughput while eliminating calibration curves. Targeted MRM analysis of neat plasma enables simultaneous detection of ~700 light/SIL peptide pairs, supporting quantification of >400 plasma proteins within a one-hour LC-MS analysis. In parallel, plasma preparation is streamlined using a one-pot (One-Tip) workflow integrating digestion, desalting, and peptide trapping within a disposable Evotip. Samples are processed and loaded directly onto C18 material for proteolysis, solid-phase extraction, and on-column injection, reducing variability and yielding reproducible identification from neat plasma (>325 proteins; median CV <20%), with ~75% peptide overlap with the SysQuan panel. SysQuan enables affordable, scalable proteome quantification.