Poster abstracts
Poster number 69 submitted by Ariana Shannon
Generating fit-for-purpose targeted mass-spectrometry based proteomics assays from a global proteome catalog.
Ariana E. Shannon (Ohio State Biochemistry Program, Pelotonia Institute for Immuno-Oncology), Yi Wang (Molecular, Cellular and Developmental Biology, Pelotonia Institute for Immuno-Oncology), Amanda B. Hummon (Ohio State Biochemistry Program, Chemistry and Biochemistry), Brian C. Searle (Ohio State Biochemistry Program, Chemistry and Biochemistry, Pelotonia Institute for Immuno-Oncology)
Abstract:
CD8+ T cells are critical players for exerting a protective immune response against malignancies. Naive T cells can differentiate into effector T cells, which exert a cytolytic role against cancer in a tumor microenvironment. Over time, effector T cells receiving constant stimulation begin to lose cytotoxic abilities and die off. The activation of CD8+ T cells is well studied by mechanisms such as antigen specific TCR presentation, however, the underlying biology that led to exhaustion remain elusive partially due to the difficulty in studying exhaustion. Current methods of studying T cell exhaustion use flow activated cell sorting (FACS) to acquire single cell protein-level data, and bulk or single cell RNA-seq data. Acquiring global proteomics data for bulk T cells is challenging, as global proteomics typically requires hundreds of thousands of cells. We have generated both global and targeted methods of analyzing T cell populations with as few as 500 cells per injection to perform mass spectrometry-based proteomics.
T cell exhaustion in cancer is difficult to study as the cells are both small in diameter, and low abundant in the tumor microenvironment. Transcriptomic data, which requires lower sample amount compared to proteomics, is typically used to study exhaustion due to the low numbers of CD8+ cells present in mouse models but does not necessarily represent true protein expression levels. To determine the minimum cell number required for proteomics measurements, effector T cell peptides were prepared in decreasing numbers (100k, 50k, 10k, 1k, 500). We detected several well-characterized peptides such as DAALMVTNDGATLIK from Cct2, which was regularly measured in 500 cell samples, and had an LoQ of approximately 1500 cells. However, most exhaustion-related peptides have LoQs between 10k and 50k cells. We will discuss estimations of additional figures-of-merit using DIA data and a new computational tool to construct targeted assays from selected proteins of interest. We will demonstrate a computer scheduled exhaustion-specific assay to quantify protein fingerprints in progenitor, acute and chronically exhausted T cells, and contrast those results with single-cell FACS data.
Keywords: proteomics, systems biology, mass spectrometry