{
  "abstract": "Background Multiplexed protein analysis in native cellular contexts holds great promise for uncovering the composition, interactions, and functions of distinct cell types in complex biological systems. However, current multiplexed protein imaging technologies often face limitations in target flexibility, detection sensitivity and sample integrity that involve technically demanding protocols. To address these issues, we have developed an ultra-sensitive, multiplexed in situ protein profiling approach that combines off-the-shelf antibodies and lectins with the newly developed Cleavable Fluorescent Probe (CFP™) cleavable TSA fluorophores.Methods Target proteins are recognized by primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase (HRP), which catalyzes the covalent deposition of CFP fluorophores on or close to the target proteins. After imaging, the fluorophores are chemically cleaved, and antibodies stripped, enabling subsequent rounds of staining, imaging, cleavage, and stripping. This approach allows for the interrogation of many targets in individual cells while maintaining spatial resolution and sample integrity.In addition to protein analysis, glycan modifications can also be profiled using this approach. Glycan modifications also play critical roles in cell function in both healthy and diseased states, from controlling proliferation to modulating the immune response. Lectins are glycan-binding proteins that recognize specific glycan structures. Their glycan-specific nature makes them important tools to profile, characterize, and capture the complexity of glycans in biological systems.We demonstrated the compatibility of CFP cleavable TSA fluorophores with lectins developed by Vector Laboratories. This assay facilitates the exploration of major glycan motifs including sialylation, fucosylation, and galactosylation, for characterizing glycan distributions in target specimens.We also utilized a novel proximity-based detection technology to investigate changes in protein glycosylation from healthy to diseased brain tissues. This tool enriches biomarker discovery and therapeutic targeting, thus emphasizing the transformative potential of glycosylation-focused research in advancing precision medicine. Results and Conclusions Our novel CFP technology enables a simplified and flexible workflow using off-the-shelf antibodies and lectins for investigating numerous targets on the same tissue section. Our results enabled us to simultaneously explore the altered protein expression and glycosylation states in specimens across various disease indications. In addition, we utilized in situ Proximity Ligation Assay to explore the glycosylation changes of specific neural cell markers in Alzheimer’s Disease brain tissues. This new data will add to the growing understanding of the impact of protein glycosylation on brain pathology.",
  "authors": [
    {
      "affiliations": [
        "Arizona State University, Tempe, AZ, USA"
      ],
      "name": "Nishinki Muthumuni"
    },
    {
      "affiliations": [
        "Arizona State University, Tempe, AZ, USA"
      ],
      "name": "Jia Guo"
    },
    {
      "affiliations": [
        "Spatomics LLC., Guilford, CT, USA"
      ],
      "name": "Dana Ashworth"
    },
    {
      "affiliations": [
        "Spatomics LLC., Guilford, CT, USA"
      ],
      "name": "Jing Zhou"
    },
    {
      "affiliations": [
        "Spatomics LLC, Newark, CA, USA"
      ],
      "name": "Xiaoshan Wang"
    },
    {
      "affiliations": [
        "Vector Laboratories, Inc. (Vector Labs), Newark, CA, USA"
      ],
      "name": "Shuhui Chen"
    },
    {
      "affiliations": [
        "Vector Laboratories, Inc. (Vector Labs), Newark, CA, USA"
      ],
      "name": "Erika Leonard"
    },
    {
      "affiliations": [
        "Vector Laboratories, Inc. (Vector Labs), Newark, CA, USA"
      ],
      "name": "Shamali Roy"
    }
  ],
  "title": "1309 Novel tools for spatial profiling of protein expression and glycosylation in brain pathology",
  "uid": "b74d29cd-504b-5814-a505-6e4e4c3d399a"
}