MSACL 2024 Industry Workshop Presentation *This Podium Presentation is occuring in the context of an Industry Workshop that starts at the time below. Its actual start time may be up to 40 minutes later, depending on order of presentation if there are multiple presentations planned by the workshop host. Workshop Host: Agilent Technologies Day: Tuesday March 19 Time: 12:15* Location: San Carlos 3 (Marriott > Mezzanine | Stairs from Lobby or SkyBridge from Conference Ctr)

A Novel Strategy utilizing SIL mouse reference material from different tissues and body fluids enabling Absolute Quantitation Of Proteins in human tissues
	Christoph Borchers, PhD 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.

Mouse is the most widely used animal model for studying human health and disease due to its size, ease of breeding, and the natural occurrence of conditions mimicking human pathology. For the past several years, my laboratory has been designing and validating multiple reaction monitoring mass spectrometry (MRM-MS) assays, and we currently have assays for the accurate quantitation of more than 2000 unique proteins in 20 distinct murine tissues and organs. The process of developing assays for accurate quantitation involves the synthesis of stable-isotope labeled (SIL) standard peptides, which can be costly and time consuming. To circumvent this problem, we have been working on a new approach – a stable-labeled mouse.
General concept of this new SysQuan approach involves, first, the generation of SIL mouse reference material is generated for different tissues and bodyfluids. Then, the concentrations of the SIL peptides are determined by comparison with reference concentrations of unlabeled peptides that are less expensive to synthesize. When a mouse tissue digest is mixed with the reference SIL mouse tissue digest, labeled and unlabeled peptides appear as doublets. After the concentrations of the peptides have been determined in the SIL mouse reference materials, they can be used as internal standards to absolutely quantify the mouse proteins in the tissue.

Because of the large number of homologous proteins between mouse and human, this approach is also applicable to the accurate quantitation of proteins in human tissues. Peptides shared between mouse and human appear as doublets when mixing human and reference SIL mouse tissues. 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 and in a fractionated run that number increases to more than 650 proteins. We expect to be able to increase the number of quantified proteins to more than 1000 by using the Evosep One on-line connected to an Agilent 6495C.