# Nanoliter-scale Affinity Purification Mass Spectrometry for Small Numbers of Hair Cells

> **NIH NIH R21** · BATTELLE PACIFIC NORTHWEST LABORATORIES · 2021 · $197,168

## Abstract

PROJECT SUMMARY/ABSTRACT
Hair cells are the sensory cells of the inner ear that carry out the essential function of mechanotransduction
evoked by sound and head movement. It is fundamentally important to characterize global protein expressions
and their interactions in hair cells in order to understand the molecular mechanism. However, one of the
greatest challenges in protein characterization is the low number of hair cells presented in each inner-ear
organ, which urges us to develop sensitive analytical approaches. To this end, our labs develop a microfluidic
sample preparation platform, termed as nanoPOTS (nanodroplet processing in one pot for trace samples), for
proteomics analysis of low-input biomaterials by downscaling processing volumes to <200 nL. While
nanoPOTS is well demonstrated to identify and quantify protein expression from single hair cells, it informs
nothing on how proteins interact with each other to implement their functions. The overall objective of this
project to develop a sensitive nanoliter droplet-based affinity purification with mass spectrometry (nanoAP-MS)
platform to identify protein interacting partners using fewer than 1000 hair cells isolated from utricles or
cochleas of the mouse ear. The central hypothesis is that the overall sensitivity of AP-MS assay can be
significantly improved by performing affinity purification in nanoliter droplets. Theory suggests this hypothesis
should be correct because: 1) Improved protein concentrations by lysing cells in nanoliter volumes will improve
protein-bead binding efficiency; 2) Reducing the amounts of affinity beads will reduce non-specific binding,
which can otherwise dwarf specific binding; and 3) improved LC-MS will provide sufficient analytical sensitivity
to measure low abundance proteins. The central hypothesis will be tested by pursuing two specific aims: 1) To
establish a nanoliter droplet-based AP-MS workflow; and 2) To apply this workflow for identification of MYO7A
and GIPC3 binding partners in mouse hair cells. We expected the proposed nanoAP-MS platform will increase
sensitivity by a factor of 103 or more and allow us to characterize low-abundance protein interaction partners.
This research is highly innovative because the nanoAP-MS platform will be the first of its kind to reliably
measure protein-protein interactions using a small number of primary cells isolated from physiological
environment, including animal models or human biopsies. Statement of Impact: As AP-MS has emerged as
powerful technology to discover protein interaction partners and establish protein-protein-interaction networks,
the nanoAP-MS technology will enable to examine important protein-protein interactions in small numbers of
cells isolated by micropipette, FACS, or laser-capture microdissection, or to examine exceptionally low-
abundance interactions like those present in the hair cell's mechanotransduction complex.

## Key facts

- **NIH application ID:** 10284899
- **Project number:** 1R21DC019753-01
- **Recipient organization:** BATTELLE PACIFIC NORTHWEST LABORATORIES
- **Principal Investigator:** Peter Gordon Barr-Gillespie
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $197,168
- **Award type:** 1
- **Project period:** 2021-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10284899

## Citation

> US National Institutes of Health, RePORTER application 10284899, Nanoliter-scale Affinity Purification Mass Spectrometry for Small Numbers of Hair Cells (1R21DC019753-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10284899. Licensed CC0.

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