Session TOD. There are 4 abstracts in this session.


Clinical Potential of Targeted Mass Spectrometry-based Proteomics in Personalized Oncology

Mandy Paulovich

Personalized oncology aims to match each patient to a specific therapy based on the molecular characteristics of their tumor. Currently, tumor DNA is sequenced, and genomics reports are given to physicians on tumor boards to help select targeted therapies for patients. While this approach has found success in extending the lives of subsets of patients, many patients do not respond to the selected therapy, and even those who do initially respond have a high chance of recurring as resistant disease. Therefore, a deeper, more comprehensive readout of tumor biology is required in order to predict tumor phenotype with respect to drug response. The majority of molecularly targeted therapies (e.g., kinase inhibitors, poly(ADP- ribose) polymerase (PARP) inhibitors, and therapies targeting immunomodulatory proteins) do not directly target the cancer genome but rather target proteins in cancer cells or the microenvironment. Thus, understanding and quantifying the expression of target proteins and their network throughout all phases of personalized oncology, from drug development to patient selection, are critically important. This presentation will discuss the added value of proteogenomics over the current genome-driven approach to the clinical characterization of cancers and summarize current efforts to incorporate targeted proteomic measurements based on selected/multiple reaction monitoring (SRM/MRM) mass spectrometry into the clinical laboratory to facilitate clinical proteogenomics. See also: Nat Rev Clin Oncol. 2019 16(4):256-268. PMID: 30487530
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Expression and Turnover of Proteoform in Cancer Aneuploidy

Yansheng Liu

Chromosome- or chromosome arm- scale DNA copy number alternations (CNAs) are called aneuploidy, which is a near-universal characteristic of human cancers. Gene expression analyses such as proteomic measurement will play a key role in understanding the effect of aneuploidy in cancer. The term 'proteoform' is now used to designate different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts, and post-translational modifications. We will present our work using data independent acquisition mass spectrometry (DIA-MS), pulse-chase stable isotope-labeled amino acids in cells (pSILAC) approach, and genome-wide correlation analysis for quantifying both abundance and turnover rate of proteins in aneuploidy models. We will further discuss the results of two major proteoforms, namely alternatively spliced protein isoform groups and protein phosphorylation, and reveal their genetic and non-genetic quantitative determinants in aneuploidy.
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Multiplex targeted MRM mass spectrometry for quantification of immunomodulatory proteins in tissue and plasma

Jeffrey Whiteaker; Lei Zhao; Richard Ivey; Regine Schoenherr; Jacob Kennedy; Amanda Paulovich
Fred Hutchinson Cancer Research Center, Seattle, WA

Quantifying the many immuno-modulatory proteins in the “cancer-immunity cycle” is critical for both patient selection and development of novel immunotherapies. Conventional technologies to measure proteins (IHC, ELISA, Western blot, Flow Cytometry) are often semi-quantitative, not multiplexable, feature poor specificity, and are difficult to develop.  Due to these limitations, investigators have turned to mRNA-based measurements to infer protein expression of immuno-modulatory factors. However, as has been unequivocally demonstrated for human breast, colorectal, and ovarian cancers (Pubmed IDs: 27251275, 25043054, 27372738), RNA levels can be poor predictors of protein levels or activity. Thus, direct quantification of proteins is desirable.


As part of the National Cancer Institute’s Cancer Moonshot, we are developing >300 multiplex assays for quantifying immunomodulatory proteins in tissue and plasma using targeted multiple reaction monitoring mass spectrometry (MRM-MS). MRM-MS complements traditional immunoassays by enabling highly specific, precise, harmonizable quantification of proteins in biospecimens, even at high multiplex levels.

As proof-of-concept, we will present a novel multiplex assay for quantification of 47 immuno-modulatory proteins in plasma and/or tumor tissue. The technology uses immunoaffinity enrichment coupled with MRM-MS (immuno-MRM assay). Assay bioanalytical validation was conducted in accordance with industry standards for use in FFPE tissue and plasma matrices and included determination of limits of detection, linear range of response, intra-day and inter-day repeatability, stability of analytes in sample processing, and characterization of reproducibility of endogenous analyte measurement. The mean linear range was over three orders of magnitude with median intra- and inter-day CVs less than 10%. The characterized assays are being applied to a panel of tumor specimens from a variety of cancer types to document detection and determine minimum sample requirements. Additional assay panels are under development to provide measurements of up to 350 immunomodulatory proteins, including signaling in the T-cell receptor network.

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Excellent sensitivity through excellent recovery – ERLIC for absolute quantification of low abundant protein phosphorylation events in cancer patient tissue

Stefan Loroch; Albert Sickmann
ISAS, Dortmund, Germany

Quantification of low abundant phosphorylation events from minute amounts of sample is a prerequisite for establishing phosphoprotoemics workflows in the clinics circumventing costly generation of antibodies. However, workflows need excellent recovery to overcome problems with low phosphoprotein stoichiometry and the limitations of sample amount (e.g. biopsies). Here we demonstrate that Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) allows improved analyte recovery and grants access to nearly all phosphopeptides in a digest. Consequently, we employed ERLIC enrichment of low abundant protein phosphorylation events in minute amounts of tissue from colon cancer patients.

To assess quantitative losses during enrichment, we spiked a “heavy”-labeled phosphopeptide-enriched fraction into an unlabeled-digest followed by another round of enrichment. Phosphopeptide ratios reflected the recovery of each phosphopeptide.

In case of TiO2-affinity purification, quantification of 1,800 different phosphopeptides revealed an average recovery of 38 ± 2% with poor recovery rates (< 10%) for a large portion of short and basic phosphopeptides. In contrast, ERLIC demonstrated a 1.7-fold higher recovery with 65 ± 15%, as determined by quantification of 1,100 different phosphopeptides. Most notably, recovery was independent of physicochemical peptide properties rendering ERLIC an excellent method for efficient purification of nearly any phosphopeptide from a digest. Since antibody-based detection repeatedly failed to work, we applied an ERLIC-based targeted assay to quantify S125 phosphorylation levels of PHD2 (mediating Hif1α degradation) in healthy and cancerous tissue of 10 colon cancer patients. PRM analysis revealed fairly stable PHD2 expression levels (358 ± 154 amol/µg) with phosphorylation levels of 2.5 ± 0.9% in all healthy tissue samples. Surprisingly, we detected diminished S125 phosphorylation levels in cancerous tissue of all 10 patients down to 0.9 ± 0.4% (p ≤ 0.001), even though total PHD2 expression remained unchanged. Subsequent, biochemical validation revealed a concomitant elevation of Hif1α expression levels rendering S125 an “on/off-switch” for PHD2 activity.

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