PXD052416

PXD052416 is an original dataset announced via ProteomeXchange.

Title	Chromatography without a Binary Pump: Smoothed Step Gradients Enable Reproducible, Sensitive, and Low-Cost Nanoflow Separations
Description	Mass spectrometry-based analysis of trace analytes, such as the single cell proteome, benefit from operation at low flow rates (i.e., <50 nL/min). However, the standard high-pressure binary pumps needed to achieve these flow rates are not commercially available, requiring splitting of the gradient flow to achieve low-nanoliter-per-minute flow rates, which wastes solvent and can lead to flow inconsistencies. To address this, we have created a new method for creating gradients by combining plugs of mobile phase of increasing solvent strength together in a column and then using Taylor dispersion to form the desired smooth gradient profile. Additionally, our method dramatically reduces costs, as only a single isocratic high-pressure pump is required. Following optimization of gradient shapes using UV absorption for both 10- and 20-min active gradients, we measured 200 pg injections of HeLa digest on a timsTOF Ultra mass spectrometer in DIA mode. We then investigated differences in protein expression between single cells originating from two different colonies of ATG-knockout HeLa cells. Thousands of proteins were quantified, and a potential mechanism explaining differential immune responses of these two colonies upon exposure to viral DNA treatment was determined.
HostingRepository	PRIDE
AnnounceDate	2025-05-07
AnnouncementXML	Submission_2025-05-06_19:49:19.154.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: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	timsTOF Ultra

Revision	Datetime	Status	ChangeLog Entry
0	2024-05-20 15:20:24	ID requested
⏵ 1	2025-05-06 19:49:20	announced

10.1016/j.mcpro.2024.100880;

Webber KGI, Huang S, Lin HL, Hunter TL, Tsang J, Jayatunge D, Andersen JL, Kelly RT, Gradient-Elution Nanoflow Liquid Chromatography Without a Binary Pump: Smoothed Step Gradients Enable Reproducible, Sensitive, and Low-Cost Separations for Single-Cell Proteomics. Mol Cell Proteomics, 23(12):100880(2024) [pubmed]

submitter keyword: Liquid Chromatography, Gradient Formation,Single-Cell proteomics

Ryan T. Kelly
contact affiliation	Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, United States of America
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/2025/05/PXD052416

PRIDE project URI

• PRIDE
  1. PXD052416
     1. Label: PRIDE project
     2. Name: Chromatography without a Binary Pump: Smoothed Step Gradients Enable Reproducible, Sensitive, and Low-Cost Nanoflow Separations