This group is utilizing a two-step process that identifies and characterizes selected proteins within cells of human tissue, which will then result in highly validated and renewable affinity reagents.

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).

Eventually there will be 30 affinity reagents (20 simple and 10 smart rABs that recognize PTMs) and hundreds of characterized proteoforms to key targets. 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 probes. These will be applicable to immunofluorescence by confocal microscopy, imaging CyTOF (MIBI), and DNA-barcoded antibodies to name a few. The stringent process of development and validation represents a general approach to better map both the chemical space of proteins and their spatial distribution in tissue at cellular and sub-cellular resolution. The combined platform focuses on the biologically active forms of proteins (proteoforms) and the PTMs decorating them that maintain health and wellness in the human body.