Dataset Information

Chromatography without a Binary Pump: Smoothed Step Gradients Enable Reproducible, Sensitive, and Low-Cost Nanoflow Separations

ABSTRACT: 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.

INSTRUMENT(S): timsTOF Ultra

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ryan Kelly

LAB HEAD: Ryan T. Kelly

PROVIDER: PXD052416 | Pride | 2025-05-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	10min-report.gg_matrix.tsv	Tabular
	10min-report.log.txt	Txt
	10min-report.pdf	Pdf
	10min-report.pg_matrix.tsv	Tabular
	10min-report.pr_matrix.tsv	Tabular

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Publications

Gradient-Elution Nanoflow Liquid Chromatography Without a Binary Pump: Smoothed Step Gradients Enable Reproducible, Sensitive, and Low-Cost Separations for Single-Cell Proteomics.

Webber Kei G I KGI Huang Siqi S Lin Hsien-Jung L HL Hunter Tyler L TL Tsang Jeremy J Jayatunge Dasun D Andersen Joshua L JL Kelly Ryan T RT

Molecular & cellular proteomics : MCP 20241112 12

Mass spectrometry-based proteome profiling of trace analytes including single cells benefits from liquid chromatography separations operated at low flow rates (e.g., <50 nl/min). However, high-pressure binary pumps needed to achieve such flow rates are not commercially available, and instead require splitting of the gradient flow to achieve low-nanoliter-per-minute flow rates. Gradient flow splitting can waste solvent and lead to flow inconsistencies. To address this, we have developed a method ...[more]

PMID: 39536954

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