MSACL 2024 Abstract

A Novel Strategy For Absolute Quantitation Of Human Proteins

Vincent R. Richard(1), Robert Popp(2), Rene Zahedi(3-6), Yassene Mohammed(1,7), Christoph H. Borchers(1,8-10*)
(1). Segal Cancer Proteomics Centre, Lady Davis Institute for Medical Research, Jewish General Hospital and McGill University, Montreal, QC H3T 1E2, Canada; (2). MRM Proteomics Inc, Montreal, QC H2X 1Y4, Canada; (3). Manitoba Centre for Proteomics and Systems Biology, Winnipeg, MB R3E 3P4, Canada; (4). Department of Internal Medicine, University of Manitoba, Winnipeg, MB R3E 3P4 , Canada; (5). Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB R3E 0J9, Canada; (6). CancerCare Manitoba Research Institute, Winnipeg, MB R3E 0V9, Canada; (7). Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; (8). Gerald Bronfman Department of Oncology, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC H3T 1E2, Canada; (9). Division of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, Canada; (10). Department of Pathology, McGill University, Montreal, QC H3A 2B4, Canada; *…presenting author

Christoph Borchers, PhD (Presenter) Jewish General Hospital, McGill University Montreal, QC, Canada

Presenter Bio: Dr. Christoph Borchers is recognized as a pioneer and leading figure in the development of mass spectrometry-based methods for protein quantification using Multiple Reaction Monitoring (MRM). He has also published more than 300 peer-reviewed papers in scientific journals, and is the founder and director of the McGill-Lady Davis Institute Integrated Proteomics Program at the Jewish General Hospital, McGill University, where he is currently a full professor in the Department of Oncology. His research is centred around the improvement, development, and application of proteomics and metabolomics technologies, with a major focus on quantitative targeted proteomics for clinical diagnostics, as well as new mass-spectrometry-based techniques for structural proteomics.
Dr. Borchers received his BS, MSc, and PhD degrees from the University of Konstanz, Germany in 1996. After his post-doctoral training and employment as a staff scientist at NIEHS/NIH/RTP, NC and he became the director of the Duke–UNC Proteomics Facility and held a faculty position at the UNC Medical School in Chapel Hill, NC (2001-2006). From 2006 to 2019, he was a Professor in the Department of Biochemistry and Microbiology, and Director of the University-Genome British Columbia Proteomics Centre at the University of Victoria, British Columbia, Canada, where he held the Rix BC Leadership Chair in Biomedical and Environmental Proteomics.
Dr. Borchers is also involved in promoting proteomics research and education through his involvement with HUPO (International Council Member), the British Columbia Proteomics Network (Executive Committee Member, past Scientific Director) and the Canadian National Proteomics Network (Member, past VP External and Chair of the Board of Directors). He is also a Fellow of the Canadian Academy of Health Sciences.

Relevant Financial Disclosures (within past 24 months, reported on Feb 25, 2022) Grant/Research Support Genome Canada Committee/Board/Advisory Board MRM Proteomics Stock/Bonds MRM Proteomics, Molecular You Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
The "gold standard" for quantitative proteomics is absolute quantitation using targeted mass spectrometry (MS) with triple quadrupole MS and stable-isotope-labeled standard (SIS) peptides. Because of the high cost of SIS peptides -- most proteomics laboratories are forced to focus on non-targeted discovery for the relative quantitation of the largest possible number of proteins. However, because of the lack of internal standards, relative quantitation is typically (i) confined to samples that are processed and analyzed together, and (ii) lacks the precision to confidently identify small but biologically relevant changes in protein expression. The cost of these SIS peptides has, thus far, precluded the use of proteomics in large clinical studies, or in large-scale or population-wide studies where 1000s of samples often need to be measured across multiple centres and countries. As a consequence, such studies have been restricted to the use of genomics methods and cannot leverage the power of additional phenotypic and information-rich proteomics.

For absolute quantitation, the amount of each isotopically labeled mouse peptide is determined by using non-isotopically labeled peptides which can be synthesized at very low cost in a high throughput manner.

We have developed the methods and standard operating procedures that allow 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).

METHODS:
This method is based on the use of body fluids and tissues from stable-isotope (i.e., 13C and 15N) labelled (SIL) mice. By analyzing these samples, we can identify surrogate peptide sequences that are identical between mouse and human proteins. Then, using cost-effective 'light' (i.e., unlabelled) synthetic peptide standards corresponding to the native peptides (NAT), we will determine the concentrations of these peptide sequences in the SIL mouse reference materials. 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).

RESULTS:
We have calculated that 13,926 proteins of the human proteome (based on 146097 tryptic peptides of length 7 to 25 amino acid) can be theoretically 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. Approximately 3000 human proteins are predicted not to have tryptic peptides suitable for quantitation. However, using alternative enzymatic digestion procedures will allow the targeting of these proteins and increase the coverage of quantifiable proteins.

Our preliminary data shows that, using this novel and unique approach, which we call "SysQuan", it is possible to achieve absolute quantitation of hundreds of proteins in human plasma in a single non-targeted LC-MS run. In fractionated run that number increases to over 650 proteins. We expect to be able to increase the number of quantified proteins to >1000 in plasma by optimizing the LC-MS conditions and acquisition method (for example, Data Independent Acquisition (DIA vs. Data Dependent Acquisition (DDA).

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