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):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ryan Kelly

LAB HEAD: Ryan T. Kelly

PROVIDER: PXD052416 | Pride | 2025-05-07

REPOSITORIES: pride

ACCESS DATA

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

Items per page:

1 - 5 of 153

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

Similar Datasets

Project description:We highlight the leap in performance made possible with new instrumentation in the Thermo Scientific Orbitrap Astral™ mass spectrometer. Integrated alongside a high-resolution accurate mass (HRAM) Orbitrap analyzer, the instrument introduces a fast-switching quadrupole mass filter congruent with downstream speeds of ion detection, a dual-pressure ion processor cell which fragments ions in the high-pressure cell before they are moved to the low-pressure cell for further accumulation and orthogonal extraction to the Astral analyzer, and a high dynamic range detector. Altogether, ions can be manipulated in five stages of the MS at once (Orbitrap, ion routing multipole, two ion processor stages, Astral analyzer), allowing for a high degree of parallelization which enables MS/MS scan speeds of 200 Hz while HRAM MS1 full scans are synchronously acquired in the Orbitrap analyzer. To provide such fast scan speeds, the Astral analyzer is capable of single-ion detection sensitivity like a linear ion trap, all while enabling resolving powers of 80,000 at m/z 524. Here, in triplicate 7-min microflow active LC gradients on the Orbitrap Astral MS, we report 7,852 protein groups from 94,267 peptides on average. When employing 15-, 30-, and 60-min active, nano-LC gradients, triplicate experiments yield an average of 9,831, 10,411, and 10,645 unique protein groups from 195,612, 234,406, and 245,754 unique peptides, respectively. These nanoflow methods delivered a median sequence coverage of 38%, 42.4% and 44.4% across identified proteins for each respective gradient length. Our 30-min method delivered approximately 347 proteins per minute. Finally, we applied our 30-min active gradient method to analyze a CRISPR-Cas9 knockout (KO) of the mitochondrial gene MGME1 in human HAP1 cells. There, we quantified an average of 10,353 proteins across three technical replicates and find 449 significantly up- or down-regulated proteins relative to wild-type (WT) HAP1 proteins. Proteins from the same sample quantified using a 61-min active gradient Orbitrap Ascend DIA method show strong fold-change correlations to and gene ontology (GO) term agreement with our results.Building upon the immense progress of the mass spectrometry and proteomics communities of the past ten years, here we report alongside other recent works, that the one-hour human proteome is now within reach.

Project description:Recent advances in mass spectrometric (MS) instruments resulting in high mass resolution and high duty cycles have allowed of very deep coverage of proteomes. However, even with state of the art, high duty cycle instruments, the number of proteins identified with a conventional single liquid chromatography-tandem MS (LC/MS/MS) analysis is typically limited and fractionating protein samples prior to LC/MS/MS analysis is crucial for increasing both the analytical dynamic range and proteome coverage. In order to achieve near comprehensive identification of proteomes two-dimensional (2D) chromatography has been an invaluable tool. This first dimension is typically performed with µL/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range while the second dimension is typically performed with nL/min flow rates and capillary columns with small inner diameters. Typically, reverse phase liquid chromatography (RPLC) provides high peak resolution of peptides and achieves higher peak capacities than strong cation exchange chromatography (SCX) due to the faster chromatographic partitioning. Another advantage of RPLC is that the mobile phases used generate much cleaner samples for downstream analyses, while the incorporation of a desalting step for SCX in order to eliminated the high salt concentrations that can significantly decrease the analytical sensitivity of the MS analysis due to ionization problems. When operated at widely different pH values (e.g., 1st dimension pH 10 and 2nd dimension pH 3) 2D RPLC-RPLC provides separation orthogonality comparable to that of the combination of SCX-RPLC. The difference of separation selectivity between the low and high pH (HpH) RPLC comes from the changes in the charge distribution within peptide chains upon altering pH of the eluent. Herein we describe a method in which tryptic peptides are separated in the first dimension by HpH-RPLC and fractions are collected every 90 s over an 80 min gradient. For the second dimension, each of these fractions is individually run by the standard low pH RPLC method. The implementation of the HpH pre-fractionation allows for short second dimension gradients for bottom up LC/MS/MS and yields very deep (e.g. thousands of identifications) protein coverage at reasonable measurement time.