SysQuan: Repurposing SILAC Mice for the Cost-Effective Absolute Quantitation of the Human Proteome

Abstract

Relative quantitation, used by most mass spectrometry-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 mass spectrometry-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 over time. However, high cost 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, of which 73,901 with lysine on the c terminus. 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 postacquisition 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.

Keywords: SILAC mice; absolute quantitation; multiplexing; stable isotope labeling; targeted and untargeted quantitation.

Graphical abstract

Fig. 1

SysQuan feasibility workflow exemplified for liver tissue. Heavy mouse (blue) and light human (red) liver tissue were homogenized, proteins extracted, and pooled 1:1 (wt/wt). Proteolytic digestion with trypsin was performed using SP3, followed by offline reversed phase fractionation at pH 10.0. Fractions were analyzed by LC-MS/MS and light/heavy doublets representing SysQuan-quantifiable peptides were identified. LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Fig. 2

SysQuan human proteome coverage.A, overlap of in-silico-generated tryptic human and mouse peptides. B, overlap of in-silico-generated tryptic human and mouse peptides with C-terminal lysine. C, based in this overlap, 68% of all human proteins in UniProtKB have shared peptides with mouse proteins, and 60% share peptides with C-terminal lysine and are therefore theoretically quantifiable using SysQuan. D, coverage and quantifiable share of the Human Liver Atlas (HLA), and (E) the Human Plasma Proteome Project (HPP) reference proteomes.

Fig. 3

Absolute quantitation of metabolomic pathway proteins.A, overall workflow. B, STRING protein/protein interaction network. C, correlation of SysQuan-derived concentrations and iBAQ-derived estimates for protein levels in proteomicsDB. iBAQ, intensity-based absolute quantitation.

Fig. 4

Agreement ofquantitationbetween two sites of measurement with different sample preparation and instrumentation.A, regression between the two analysis on two different liquid chromatography systems as well as mass spectrometers showing that most of the Log₂ ratios are centered around 0. B, Bland–Altman analysis showing an agreement of 95.35% between two measurements. LoA, limit of agreement; Mean Diff, mean difference.