MassIVE MSV000098998

Partial Public PXD067958

Definitive benchmarking of DDA and DIA for host cell protein analysis on the Orbitrap Astral in a regulatory-aligned framework

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Description

Host cell proteins (HCPs) are critical quality attributes in biotherapeutics that require accurate, specific, and comprehensive quantification. Mass spectrometry (MS)-based workflows are increasingly adopted to overcome the coverage and specificity limitations of immunoassays. This study benchmarks the performance of the Orbitrap Astral mass spectrometer for label-free HCP analysis, comparing data-dependent acquisition (DDA, Top80) and data-independent acquisition (DIA, 4 m/z non-staggered windows) modes. We applied a statistically rigorous framework integrating a stable isotope-labeled HCP mixture for traceable quantification, entrapment-based empirical false discovery proportion estimation, deterministic protein inference, and stratified bootstrapping. Both acquisition modes demonstrated exceptional quantitative fidelity. DIA outperformed DDA in identifications, yielding 45% more proteins and 68% more peptides. Hierarchical Bayesian modeling revealed superior differential linearity in DIA (mean slope 1.0) compared to DDA (slope 0.8). Stratified bootstrap analysis confirmed linearity and accuracy across the dynamic range, with DIA achieving lower limits of quantification (0.6 ppm) versus DDA (1.6 ppm). While both workflows reliably quantified most high-risk HCPs, DIA provided expanded proteome coverage and enhanced fold-change precision. These findings validate the Orbitrap Astral as a high-fidelity platform for HCP analysis in both modes and establish a defensible, regulatory-aligned MS-based framework for routine use in quality control environments. [doi:10.25345/C5T14V28X] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: HCP ; DDA ; DIA ; Orbitrap Astral ; Host cell protein ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Somar Khalil, GSK, Belgium
Submitting User: SomarKhalil
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Identification Results
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Quantification Results
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Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

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Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

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Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

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Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

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Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

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Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
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