# Droplet sample preparation

## Introduction

Mass spectrometry methods have enabled quantifying thousands of proteins at the single cell level. These methods open the door to many biological opportunities, such as characterizing heterogeneity in the tumor micro-environment, signaling pathways driving stem cell differentiation, and intrinsically single-cell processes, such as the cell division cycle. To further advance single-cell MS analysis, we developed an automated nano-ProteOmic sample Preparation (nPOP). nPOP uses piezo acoustic dispensing to isolate individual cells in 300 picoliter volumes and performs all subsequent preparation steps in small droplets on a hydrophobic slide. This allows massively parallel sample preparation, including lysing, digesting, and labeling individual cells in volumes below 20 nanoliters.

## Application

Single-cell protein analysis using nPOP classified cells by cell type and by cell cycle phase. Furthermore, the data allowed us to quantify the covariation between cell cycle protein markers and thousands of proteins. Based on this covariation, we identify cell cycle associated proteins and functions that are shared across cell types and those that differ between cell types.

## Raw Data from experiments benchmarking nPOP

• MassIVE Repository:

## Processed Data from experiments benchmarking nPOP

• Peptides-raw.csv
• Peptides x single cells at 1% FDR. The first columns list the peptide sequences and each subsequent column corresponds to a single cell. Peptide identification is based on spectra analyzed by MaxQuant and is enhanced by using DART-ID to incorporate retention time information. See Specht et al., 2019 for details.

• Proteins-processed.csv
• Proteins x single cells at 1% FDR, imputed and batch corrected.

• Cells.csv
• Annotation x single cells. Each column corresponds to a single cell and the rows include relevant metadata, such as, cell type, measurements from the isolation of the cell, and derivative quantities, i.e., rRI, CVs, reliability. This file corresponds to Proteins-processed.csv and Peptides-raw.csv files.

• HeLa-proteins.csv
• Proteins x single cells for HeLa cells at 1% FDR, unimputed and z-scored.

• U-937-proteins.csv
• Proteins x single cells for U-937 cells at 1% FDR, unimputed and z-scored.