# Automated processing and manipulation of small samples for high throughput and ultrasensitive functional proteomics measurements

> **NIH NIH P41** · BATTELLE PACIFIC NORTHWEST LABORATORIES · 2022 · $250,326

## Abstract

Project Summary – TR&D 1
The overarching goal of the Resource is to dramatically improve on the existing proteomics paradigm by
achieving orders-of-magnitude gains in both measurement sensitivity and throughput, as well as to address
important measurement `blind spots' and shortcomings of current methods. At present, a minimum of
thousands of cells are generally required for in-depth coverage of proteins in a biological sample, precluding
many important applications involving extremely small samples, rare cell types or spatially resolved
measurements. We have recently developed a Nanodroplet Processing in One-pot for Trace Samples
(nanoPOTS) technology which, when coupled with ultrasensitive MS-based measurements, enables effective
analysis of as few as 10 mammalian cells. The technology efforts of TR&D 1 will extend this
robotic/microfluidic nanoPOTS platform to provide the `up-front' processing required to efficiently handle
ultra-small samples (extending to single cells) and deliver these samples optimally to the Structures for
Lossless Ion Manipulations (SLIM)-based ion mobility-mass spectrometry (IM-MS) platforms to be further
advanced under TR&D 2. The robotic platform will enable samples to be transferred using nanoelectrospray
ionization to the SLIM IM-MS platforms with high ionization and utilization efficiencies. The nanoPOTS
platform and workflow will be optimized to enable broad proteome coverage for sample sizes in the range of
1-1000 cells so as to enable broad proteome coverage. The efforts will also establish seamless, automated
integration of nanoPOTS with the widely used cell isolation technologies of fluorescence activated cell sorting
(FACS) and laser-capture microdissection (LCM), enabling ultra-rare cell analysis and high-resolution
proteome mapping of clinical tissues. We will also extend nanoPOTS processing to address the inherently small
sample sizes and sensitivity challenges associated with functional proteomics measurements, including
activity-based proteomics and measurements of key post-translational modifications such as phosphorylation,
using a combination of direct surface functionalization, magnetic bead-based workflows within the nanowells
and microcolumn fractionation to enrich and process functional protein subpopulations. We aim to reduce
required sample sizes for such functional measurements by more than 100-fold. The efficient processing and
greatly reduced sample losses enabled by the nanoPOTS platform will complement the large gains in
throughput and sensitivity afforded by TR&D 2 and its fast and high-resolution gas-phase separations and
manipulations in SLIM. The workflows to be developed will enable both online and offline separations (e.g.,
liquid chromatography) with SLIM IM-MS using nanoPOTS-prepared samples. The development of this
platform will occur in close collaboration with the TR&D 2 and 3 efforts and is key to most of the driving
biological projects.

## Key facts

- **NIH application ID:** 10461818
- **Project number:** 5P41GM103493-20
- **Recipient organization:** BATTELLE PACIFIC NORTHWEST LABORATORIES
- **Principal Investigator:** Ryan T Kelly
- **Activity code:** P41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $250,326
- **Award type:** 5
- **Project period:** 2003-09-15 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10461818

## Citation

> US National Institutes of Health, RePORTER application 10461818, Automated processing and manipulation of small samples for high throughput and ultrasensitive functional proteomics measurements (5P41GM103493-20). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10461818. Licensed CC0.

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