# Renewable and Specific Affinity Reagents for Mapping Proteoforms in Human Tissues

> **NIH NIH UH3** · NORTHWESTERN UNIVERSITY · 2020 · $400,000

## Abstract

Abstract
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 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. Our 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. PTM maps enabled by smart
and renewable reagents represent a high value outcome, templated here through this collaborative effort
between the Wells and Kelleher laboratories both coordinating effectively with each other and the HuBMAP
Consortium.

## Key facts

- **NIH application ID:** 10249071
- **Project number:** 5UH3CA246635-02
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** NEIL L KELLEHER
- **Activity code:** UH3 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $400,000
- **Award type:** 5
- **Project period:** 2019-09-15 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10249071

## Citation

> US National Institutes of Health, RePORTER application 10249071, Renewable and Specific Affinity Reagents for Mapping Proteoforms in Human Tissues (5UH3CA246635-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10249071. Licensed CC0.

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