What Does Next-Generation Mass Spectrometry Offer for Proteomics? A Comprehensive Platform Comparison

Area of Science:

• Proteomics and Systems Biology
• Mass Spectrometry
• Biomolecular Analysis

Background:

• Next-generation mass spectrometry platforms like Orbitrap Astral and timsTOF Ultra offer enhanced sensitivity and analytical depth.
• Previous platforms, such as Orbitrap Exploris 480, provided a baseline for comparison in proteomic studies.
• Bronchopulmonary dysplasia models in neonatal mice present a complex biological system for proteomic investigation.

Purpose of the Study:

• To compare the performance of next-generation mass spectrometry platforms (Orbitrap Astral, timsTOF Ultra) against a current-generation platform (Orbitrap Exploris 480).
• To evaluate different data acquisition strategies, including data-dependent acquisition (DDA) and data-independent acquisition (DIA), on these platforms.
• To assess the impact of enhanced proteome depth on systems biology analyses, including subcellular localization and pathway enrichment.

Main Methods:

• Comparative proteomic analysis using Orbitrap Exploris 480 (DDA/DIA), Orbitrap Astral (HR-DIA), and timsTOF Ultra (DIA-PASEF).
• Utilized neonatal mouse lung tissues from a bronchopulmonary dysplasia model (n=12).
• Analyzed proteome coverage, peptide and protein quantification, sample size requirements, subcellular compartment annotation, and reactome pathway coverage.

Main Results:

• All platforms identified approximately 4000 common proteins; DIA methods achieved 98% proteome coverage.
• Orbitrap Astral and timsTOF Ultra quantified significantly more peptides and proteins compared to Orbitrap Exploris 480 DDA.
• Next-generation platforms reduced the recommended sample size by approximately 66% and improved subcellular and pathway annotations.
• Differential expression analysis revealed more phenotype-associated proteins and enriched pathways using DIA data, without functional annotation bias.

Conclusions:

• Data-independent acquisition (DIA) on multivendor next-generation mass spectrometry platforms provides superior proteome coverage and depth.
• These advanced platforms enable more comprehensive systems biology assessments, enhancing the understanding of complex biological systems.
• The increased proteomic depth and efficiency of next-generation platforms facilitate more robust biomarker discovery and biological interpretation.