A goal of HuBMAP is to characterize the molecular complexity of differing biological tissues. To address the sensitivity limitations of existing procedures for organizing and optimizing the identification of types and amounts of specific proteins present in a biological sample—called proteomics workflows—Pacific Northwest National Laboratories (PNNL) has developed a new technology for analyzing proteins in small samples.

Called nanodroplet Processing in One pot for Trace Samples (nanoPOTS), the new method is an automated sample preparation platform that greatly expands the number of proteins that can be analyzed and microscopically imaged in small samples. NanoPOTS results in an extremely small sample volume—about 200 nanoliters—that enables ultrasensitive nanoLC-MS/MS (MS, or mass spectroscopy, fragmenting biomolecules and identifying their components chemically) with little sample loss in the MS process. Thus nanoPOTS can analyze tissue voxels—the equivalent of a pixel in a video image, but in three dimensions—generating quantitative cell-type-specific images for more than 2,500 proteins in a given sample with 100-micrometer spatial resolution. Combining nanoPOTS with nanospray desorption electrospray ionization (nano-DESI) imaging, developed by the Purdue University Transformative Technology Development (TTD) group, allows the construction of detailed spatial maps of proteins, lipids and metabolites. To obtain single cell resolution, nanoPOTS can be coupled with powerful targeted technologies such as Immunostaining with Signal Amplification by Exchange Reaction (Immuno-SABER), which amplifies the signal from an antibody attaching to a specific protein by attaching a repeated DNA sequence to the antibody that allows multiple fluorescent labels. Immuno-SABER can amplify the protein signal in tissue samples in a multiplexed and individually controllable manner.

In the proposed collaborative research project between the Purdue University and Harvard University TTDs, the teams will use nanoPOTS to uncover global proteomic changes across differing tissue sections and Immuno-SABER to provide cellular resolution and sensitivity for targets of interest. In these initial experiments the collaborators will utilize uterine tissue sections containing luminal epithelial cells, stromal cells and dispersed glandular epithelial cells. They will also use high-resolution nano-DESI imaging of lipids and metabolites together with nanoPOTS imaging of proteins to guide the target selection for Immuno-SABER experiments.