Dataset Information

LFQ Benchmark Dataset - Generation Beta: Assessing Modern Proteomics Instruments and Acquisition Workflows with High-Throughput LC Gradients - Optimized Astral dataset

ABSTRACT: Recent advancements in liquid chromatography-mass spectrometry (LC-MS) have increasingly focused on high-throughput workflows, leveraging rapid chromatographic gradients and minimal sample input to maximize proteome coverage from limited material. This shift is particularly driven by the rise of single-cell proteomics, where sensitivity and reproducibility are critical. Building on our previous benchmark dataset (PXD028735), we now present an expanded study utilizing the latest generation of LC-MS platforms optimized for high-throughput proteomics. This study features shorter LC gradients and lower sample input to address the growing need for rapid and sensitive proteome analysis. Using a standardized hybrid proteome mixture with defined ratios of Human, Yeast, and E. coli, we generated a comprehensive Data-Dependent and Data-Independent Acquisition (DDA/DIA) dataset across multiple state-of-the-art LC-MS platforms. The updated dataset incorporates the latest acquisition methodologies and extends coverage across an even broader range of data formats, including enhanced ion mobility-enabled and scanning quadrupole-based acquisitions. Our results providea detailed assessment of the impact of technological advancements and demonstrate how shortening LC gradients influence proteome coverage, quantitative precision, and data consistency across instruments

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Bart Van Puyvelde

LAB HEAD: Maarten Dhaenens

PROVIDER: PXD071205 | Pride | 2026-02-03

REPOSITORIES: Pride

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			Action	DRS
	F013347.mgf	Mgf
	F013347_Astral.mzid	Mzid
	LFQ_Astral_DIA_Optimized_DI_15min_250pg_Condition_A_REP1.raw	Raw
	LFQ_Astral_DIA_Optimized_DI_15min_250pg_Condition_A_REP2.raw	Raw
	LFQ_Astral_DIA_Optimized_DI_15min_250pg_Condition_A_REP3.raw	Raw

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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.

Dataset Information

LFQ Benchmark Dataset - Generation Beta: Assessing Modern Proteomics Instruments and Acquisition Workflows with High-Throughput LC Gradients - Optimized Astral dataset

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