Relative quantitation, used by most MS-based proteomics laboratories to determine protein fold-changes, requires samples being processed and analyzed together for best comparability through minimizing batch differences. This limits the adoption of MS-based proteomics in population-wide studies, and the detection of subtle but relevant changes in heterogeneous samples. Absolute quantitation circumvents these limitations and enables comparison of results across laboratories, studies, and longitudinally. However, high costs of the essential stable isotope labeled (SIL) standards prevents widespread access and limits the number of quantifiable proteins. Our new approach, called “SysQuan”, repurposes SILAC mouse tissues/biofluids as system-wide internal standards for matched human samples to enable absolute quantitation of, theoretically, two-thirds of the human proteome using 157,086 shared tryptic peptides. We demonstrate that SysQuan enables quantification of 70% and 31% of the liver and plasma proteomes, respectively. We demonstrate for 14 metabolic proteins that abundant SIL mouse tissues enable cost-effective reverse absolute quantitation in, theoretically, 1000s of human samples. Moreover, 10,000s of light/heavy doublets in untargeted SysQuan datasets enable unique post-acquisition absolute quantitation. SysQuan empowers researchers to replace relative quantitation with affordable absolute quantitation at scale, making data comparable across laboratories, diseases and tissues, enabling completely novel study designs and increasing reusability of data in repositories.

Absolute quantitation of metabolomic pathway proteins S-TRAP-digests of light human and SIL mouse liver were mixed 1:1 (w/w) based on BCA protein concentrations. A calibration curve was generated with 0.1, 1, 10, 100, 1000 fmol, each, of the NAT peptides for the 14 target proteins ACO1, ASS1, COX7C, DLAT, GAPDH, GOT1, HADH, NDUFA4, NDUFS4, NDUFV1, NDUFV2, SLC25A3, SUCLA2, SUCLG1 (see also Table 1 and Supplemental Table S1) in constant SIL mouse liver background. Next, human/mouse liver mix and the calibration curve were measured by LC-MRM on an Agilent 6495C mass spectrometer on-line coupled to an Agilent 1290 HPLC. Peptides were separated on an Agilent Zorbax Eclipse Plus RP-UHPLC column (2.1 x 150 mm, 1.8 µm) using a 47-min binary gradient (A: 0.1% FA; B: 100% ACN) ramping from 2-7% B in 2 min, 7%-27.6% B in 43 min, and 27.6-80% B in 2 min. A total of 930 MRM transitions were acquired for the 14 light and 14 SIL peptides using dynamic MRM and 2-min retention time windows, with minimum and maximum dwell times of 1.62 ms and 71.85 ms, respectively; the maximum cycle time was 555.59 ms (see Supplemental Data Table S2). Data was analyzed using Skyline. Calibration curves were used to calculate target analyte concentrations (fmol analyte / µg of liver tissue) in the human liver sample for a given light:heavy peak area ratio.

S-TRAP-digests of light human (BioIVT) and SIL mouse liver (Silantes) mixed 1:1 .