Session: Clinical Proteomics

Session Chair: Prof. Dr. Christoph Borchers

Immuno-multiple reaction monitoring (iMRM) for the targeted ‘absolute’ quantitation of cancer proteins in tumor tissue samples

Prof. Dr. René Zahedi, University of Manitoba
Precision Oncology (PO) has revolutionized the classification and treatment of tumors. Mostly genomic biomarkers are being used to predict targeted therapies for individual patients, which has led to spectacular successes for advanced cancers and dramatically improved outcomes for certain lethal cancers. Yet, only 13.6% of US cancer patients are eligible for targeted therapies, of which only ~52% respond. On the one hand, there are no actionable targets for many tumors, either because of tumor biology, missing data, or the lack of appropriate drugs to target identified drivers. On the other hand, in the light of complex tumor biology and tumor heterogeneity, existing biomarkers often do not provide the anticipated response rates. The low response rates of current PO can be partially attributed to a disconnect between genomics data and the actual activity of cancer signaling pathways and drug targets on the protein level, but also to the use of semi-quantitative methods to determine protein expression, such as immunohistochemistry (IHC). To improve cancer patient stratification, we have developed immuno-MS assays (iMRM) for a number of cancer-related target proteins. The combination of a quality-controlled immuno-enrichment step with robust high-flow LC-MRM, that is well-established in the clinic, allows for the robust and ‘absolute’ quantitation of selected protein targets with high sensitivity. Thus, we have developed multiplexed iMRM assays for targets such as PTEN, the main negative regulator of the PI3K/AKT/mTOR pathway, as well as members of the PD-L1 signaling axis. Our assays show a higher precision and sensitivity than existing assays based on western blot or immuno-histochemistry, while concurrently providing actual protein concentrations rather than semi-quantitative readouts. First data indicate a predictive value of the determined protein concentrations with regard to drug response or overall survival, which still needs to be validated in larger cohorts. Importantly, the standardized nature of our assays allows to perform inter-laboratory studies in order to establish robust reference concentration ranges that might be used for patient stratification.
22-Jun-2022 12:30 (30 Minutes) ICM/Hall 2

Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS

Prof. Dr. Bernhard Kuester, TU München
Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC–MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC–MS/MS using a 1?×?150?mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16?h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16?h. The system identifies >30,000 phosphopeptides in 12?h and protein-protein or protein-drug interaction experiments can be analyzed in 20?min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC–MS/MS is suitable for a broad range of proteomic applications.
22-Jun-2022 13:00 (30 Minutes) ICM/Hall 2

MALDI Mass Spectrometry Imaging in (Pre)Clinical Research

Prof. Dr. Carsten Hopf, Hochschule Mannheim
MALDI MS Imaging (MSI) has recently emerged as a key label-free and fast technology for the assessment of spatial distribution of (onco-) metabolites, N-glycans, lipids, drugs, drug metabolites in preclinical animal models of disease and in human tissue specimen. MSI provides molecular specificity information by ultra-high resolution MS, e.g. magnetic resonance MS, or trapped ion mobility MS [1]. Part 1 of the talk will present recent clinical applications of (quantitative) MSI in spatial drug and oncometabolite analysis in patient tissues [2] and highlight the use of MSI in spatial analysis of tissue enzyme function [3]. Current developments of MSI technology for (pre)clinical research will be explored in part 2 of the talk: 3D-reconstructions combined with computational analysis [4], multimodal infrared imaging-guided MSI, bioanalysis of 3D-cell culture models of disease, or the coupling of MSI with laser microdissection and LC-MS.
22-Jun-2022 13:30 (30 Minutes) ICM/Hall 2

Protein quantification assay knowledgebase and applications in pre-clinical research

Dr. Yassene Mohammed, Leiden University Medical Center
Precise multiplexed quantification of proteins in biological samples can be achieved by targeted proteomics using multiple or parallel reaction monitoring (MRM/PRM). Combined with internal standards, the method achieves very good repeatability and reproducibility enabling excellent protein quantification and allowing longitudinal and cohort studies. In order to streamline the development and validation of targeted protein assays, we have introduced MRMAssayDB1 , an integrated resource for targeted proteomics assays. The Web-based application archives the targeted proteomics assays in the community, i.e. >828 000 assays covering >51 000 proteins from 94 organisms. Our laboratory (at University of Victoria) optimized the multiplexed quantification of up to 270 proteins with internal standards in less than 10 ?l plasma. We applied these assays in clinical setting to assess the applicability and usability of such assays in preclinical research. In this work, we will provide an overview of three cohort studies, in which the results of the 45 minutes fast multiplexed targeted proteomics plasma assays outperformed traditional methods in assessing disease and health. This includes works on evaluating risk of venous thrombosis following traumatic lower-leg injury or knee arthroscopy, in prediction of venous thromboembolism in patients with colorectal cancer, and in evaluating longitudinal COVID blood profile2-6 .
22-Jun-2022 14:00 (30 Minutes) ICM/Hall 2