Proteome-Seq: Sequencing-Based Readout Of Proteomic Analytical Assay

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Long, Mariia, Alibekova

Cell functional states undergo rapid changes during development and in disease states, reflecting heterogeneity inherent to cellular systems. This heterogeneity arises from multiple factors, including environmental cues, genomic variations, epigenetic modifications, transcriptional dynamics and the vast complexity of proteome. While RNA sequencing is a workhorse of high-resolution single-cell analysis, it captures cellular states pre protein translation. Additionally, cell RNA abundance correlates poorly with protein abundance, thus explaining only fraction of protein variability within the cells. Proteins, primary drivers of cellular functions and processes, exhibit great variability in copy numbers and post-translational modifications, making them necessary albeit challenging targets for high resolution analysis. Unlike RNA, proteins can’t be amplified directly, posing a significant challenge to existing proteomics methodologies. Even cutting-edge proteomics methods often encounter trade-offs between sensitivity, target number and the ability to detect low abundance proteins within single cells. This suggests a gap in our understanding of cellular functional states when proteomic analysis methodologies do not offer resolution similar to that of RNA-seq. We aim to bridge this gap in this thesis work by introducing a novel methodology termed Proteome-seq, which leverages DNA-based encoding and next generation sequencing-based readout of protein information. This approach includes two crucial pieces of protein identity information in DNA form: Antibody ID, a DNA barcode conjugates to an antibody specific to the target of interest, and Location ID, a DNA barcode encoding spatial information within the assay medium. By leveraging the amplification capabilities inherent to DNA, we aim to improve detection of low abundance targets while enabling analysis of an extensive array of targets in high throughput, unconstrained by limitations of traditional optical readouts. Our Proteome-seq workflow starts with single cell Western blots (scWestern) which allows us to physically separate proteins in thousands of cells simultaneously by SDS-PAGE. We overcame limitations of sensitivity of traditional scWesterns by introduction of diffusive blotting of protein targets to nitrocellulose membrane. This allows us to achieve higher protein immobilization, greater detection sensitivity and enabled probing of scWesterns with antibody conjugates. We have also implemented DNA tagged antibodies for in situ protein signal amplification. After scWestern proteins are immobilized, we perform DNA-based encoding. Subsequent analysis with next generation sequencing allows to reconstruct protein signal in DNA form, ensuring minimal loss in resolution and favorable signal-to-noise ratio. Versatility of Proteome-seq approach extends beyond scWesterns, as it is compatible with various proteomic assays such as Western blotting, thereby expanding its capabilities through increased target coverage and optics-free image reconstruction. This comprehensive approach not only makes headway towards addressing challenges in the field of single-cell proteomics and paves a way toward better understanding of cell functional states through bridging analytical gap between proteomics and sequencing.

Hughes, Alex
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