PXD070201-1

PXD070201 is an original dataset announced via ProteomeXchange.

Title	High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time
Description	Mass spectrometry (MS)-based single-cell proteomics (SCP) enables proteome-wide analysis at single-cell resolution, offering insights into cellular heterogeneity, biological processes, and disease mechanisms. However, conventional nanoLC-MS workflows are constrained by long gradients and low throughput, limiting their application to large cohorts. Here, we present a multicolumn nanoLC–MS platform that achieves 5-minute separation windows with 100% duty cycles at ~100 nL/min, enabling the analysis of up to 288 single cells per day with minimal additional hardware. The system provides stable peptide separation, negligible carryover, and robust retention-time reproducibility across 2,000 consecutive injections. Over the course of this study, we successfully analyzed more than 4,000 samples with ~288 samples per day (SPD) throughput. The platform identified ~4,400 proteins per 250 pg digest injection and an average of ~3,200 proteins per single HeLa cell with maxima exceeding 4,300, which is comparable to state-of-the-art longer-gradient workflows. Quantitative benchmarking with mixed-species standards confirmed accurate measurements across ~6,000 proteins. The workflow distinguished proteomic profiles across hundreds of single cells and, importantly, profiling of RAW264.7 macrophages revealed LPS-induced markers and macrophage activation pathways. Together, these results establish a robust and scalable platform for high-throughput SCP, demonstrating the feasibility of thousands of single-cell analyses within a single study while maintaining deep proteome coverage and biological interpretability.
HostingRepository	PRIDE
AnnounceDate	2026-05-22
AnnouncementXML	Submission_2026-05-21_22:22:24.311.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ryan Kelly
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606; scientific name: Mus musculus (Mouse); NCBI TaxID: NEWT:10090;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Astral

Revision	Datetime	Status	ChangeLog Entry
0	2025-11-02 21:14:37	ID requested
⏵ 1	2026-05-21 22:22:24	announced

Dataset with its publication pending

submitter keyword: 5-min cycle time, 288 SPD,high-throughput SCP, nanoLC-MS/MS

Ryan T. Kelly
contact affiliation	Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States
contact email	ryan.kelly@byu.edu
lab head
Ryan Kelly
contact affiliation	Brigham Young University
contact email	ryan.kelly@byu.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD070201

PRIDE project URI

• PRIDE
  1. PXD070201
     1. Label: PRIDE project
     2. Name: High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time