Droplet proteomic sample preparation (nPOP)

Massively parallel sample prep method by Leduc et al, 2021

Peer reviewed article: Leduc A, Huffman RG, Cantlon J, Khan S, Slavov N Exploring functional protein covariation across single cells using nPOP Genome Biol 23, 261 10.1186/s13059-022-02817-5, Data Websites

Protocol article: Leduc A, Khoury L., Cantlon J., Khan S., Slavov N. Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP, Nature Protocols, doi: 10.1038/s41596-024-01033-8, Blog post, Protocol preprint: Leduc et al., bioRxiv 2023.11.27.568927, doi: 10.1101/2023.11.27.568927

Research Article Protocols GitHub nPOP videos

Data Websites

Introduction Resources Advantages of nPOP Applications Videos

Introduction

The nano-ProteOmic sample Preparation (nPOP) uses piezo acoustic dispensing to isolate individual cells in 300 picoliter volumes and performs all subsequent preparation steps in small droplets on a fluorocarbon-coated slide. This design enables simultaneous sample preparation of thousands of single cells, including lysing, digesting, and labeling individual cells in volumes below 20 nl.

Advantages of nPOP

nPOP can prepare thousands of single cells in a single batch
- This minimizes the # of batches and associated technical variation
nPOP uses only mass-spec compatible chemicals and volumes below 20 nl
- This results in low background and low contamination
nPOP droplet layouts are computationally programmable and thus very flexible
- Thus nPOP can be used for all multiplexed or label-free workflows without specialized consumables
nPOP uses accessible consumables and small amounts of labels
- This keeps costs down to about 10-20 cents / single cell.

nPOP uses the CellenONE instrument, which is commercially available. Without access to CellenONE, one may use mPOP, which can prepares a few hundred single cells in parallel. Both mPOP and nPOP are fully compatible with all mass-spectrometry methods described in this portal.

Applications & data

Leduc et al, 2021 used nPOP to prepare samples for SCopE2 analysis of the cell division cycle of U937 and Hela cells. Leduc et al, 2021 data website
Derks et al, 2022 used nPOP to prepare samples for plexDIA analysis of single melanoma, monocyte and pancreatic adenocarcinoma cells. Derks et al, 2022 data website
Leduc et al, 2022 used nPOP to prepare samples for pSCopE analysis of protein covariation in monocytes and melanoma cells. Leduc et al, 2022 data website
Huffman et al, 2022 used nPOP to prepare samples for pSCopE analysis of primary macrophages stimulated with LPS. Huffman et al, 2022 data website
Leduc et al, 2023 used nPOP to prepare samples for plexDIA analysis of single melanoma, monocyte and pancreatic adenocarcinoma cells. Leduc et al, 2023 data website
Khan et al, 2023 used nPOP to prepare samples for SCoPE2 analysis of single epithelial cells undergoing epithelial–mesenchymal transition. Khan et al, 2023 data website
Derks et al, 2024 used nPOP to prepare samples for plexDIA analysis of single nuclei. Derks et al, 2024 data website
Leduc et al, 2024 used nPOP to prepare samples for pSCopE analysis of single cells from mouse tissues. Leduc et al, 2024 data website

Resources and protocols

The current nPOP protocol has been published in Nature Protocols by Leduc et al.. This latest version of the protocol was created in partnership with Cellenion, and the company provides support for implementing nPOP, including required software for the protocol, which can be obtained through our partnership program. Reaching out to Josh Cantlon (j.cantlon@cellenion.com) or Andrew Leduc (leduc.an@northeastern.edu) for more details.
The original version of the nPOP protocol can be found here and the cellenONE software files for performing sample prep can be found here.
Additionally, tools for mapping single cell image data from the cellenONE to sets run for LC/MS are available here on this GitHub page.

A google group for questions and answers about nPOP.