Renewable and Specific Affinity Reagents for Mapping Proteoforms in Human Tissues

The mapping of healthy human tissue is an ongoing challenge, critical to which is understanding the cellular distribution and composition of biomolecules. Among these biomolecules, proteins are perhaps the least regularized and deeply understood, with their myriad proteoforms created within cells of distinct types. Here, we outline a new measurement framework poised to better illuminate the underlying cell types and structure defining solid tissues types in human health and wellness. We propose a two-step process that identifies and characterizes selected proteins within cells of human tissue and offers the HuBMAP Consortium highly validated and renewable affinity reagents in the process. Step 1 involves the rapid deployment of previously developed, recombinant affinity reagents (rABs) and the precise characterization of whole proteoforms in human colon tissue recognized by them. Step 2 involves the creation of “smart” probes that work on specific post-translational modifications (PTMs) in a ‘proteoform-aware’ fashion. The result will be specific, renewable and extremely validated affinity probes that are readily disseminated for use by HuBMAP Tissue Mapping Centers (TMCs). Importantly, these recombinant antibodies (rABs) will be among the most characterized of any in terms of their molecular recognition by the end of the three year granting period. This is because of the kind of low-bias readout that employs “top-down” mass spectrometry (TDMS) to interrogate whole proteoforms and their PTMs directly (no proteolysis prior to mass spectrometry). The direct deliverable of this activity will be 30 affinity reagents (20 simple and 10 smart rABs that recognize PTMs) and hundreds of characterized proteoforms to key targets of high interest to HuBMAP. The affinity reagents that emerge will be in a recombinant and renewable form (i.e., proven to express in E. coli) that are ready for use in a variety of imaging modalities through conjugation to fluorescent