SysQuan: a novel strategy for the absolute quantitation of human proteins

Introduction

The "gold standard" for quantitative proteomics is absolute quantitation using targeted mass spectrometry (MS) with triple quadrupole MS and stable-isotope-labeled (SIL) peptides. The high cost of SIL peptides has, thus far, precluded the use of proteomics in large clinical studies where 1000s of samples often need to be measured. We have developed a new method that allows the absolute quantitation of 1000s of proteins in different human biofluids and tissues at a fraction of the current cost, and which can be used for both classical targeted MS and non-targeted approaches, such as data independent acquisition (DIA). This method is based on the use of body fluids and tissues from stable-isotope (13C- and 15N) labelled (SIL) mice.

Methods

This method is based on the use of body fluids and tissues from stable-isotope labelled (SIL) mice. By analyzing the labeled mouse samples, we can identify surrogate peptide sequences that are identical between mouse and human proteins. When mixed with the corresponding human tissue/body fluids, all of the peptides that can be used for absolute quantitation will appear as doublets (light/heavy pairs). Then, using cost-effective "light" (i.e., unlabelled) synthetic peptide standards corresponding to the native peptides (NAT), we can determine the concentrations of these peptide sequences in the SIL mouse reference materials.

Results

We have calculated that 13,926 proteins of the human proteome (based on 146097 tryptic peptides 7 to 25 amino acids long) can theoretically be quantified using this approach. This means that, for the first time, the "proteome-wide" absolute quantitation can be performed, without the current limitations of quantitative MS. For those proteins without suitable tryptic peptides for quantitation, we will use alternative enzymatic digestion procedures.

We have already shown that it is possible to achieve absolute quantitation of hundreds of proteins in human plasma in a single non-targeted LC-MS run on the timsTOF. In a fractionated run using that number increases to >650 proteins.

Using high pH pre-fractionation (with 48 fractions) followed by DDA on an Evosep interfaced to a timsTOF we were able to detect 1159 proteins (covered by 3639 heavy-light peptide pairs) in plasma and 6987 proteins (covered by 48760 heavy-light peptide pairs) in liver. We are in the process of transforming our untargeted LC-MS/MS assay into a targeted LC-MRM/MS approach for absolute quantitation using the Evosep/Agilent 6495D QQQ equipment. Our first project will be the >1000 proteins detected in liver which are involved in metabolic pathways.

All of the acquired peptide doublets can later be assigned to SIL mouse concentrations, even if these are determined post-acquisition. This new SysQuan approach will be a "game-changer" for non-targeted and absolute protein quantitation as it theoretically would allow a researcher to quantify the entire human proteome.