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

Next-generation
mass spectrometry platforms (Orbitrap Astral, timsTOF
Ultra) are reshaping proteomics by enhancing analytical depth and
sensitivity. We compared these platforms against Orbitrap Exploris
480 using neonatal mouse lung tissues from a bronchopulmonary dysplasia
model (n = 12), employing four acquisition strategies:
Exploris 480 DDA/DIA, Astral HR-DIA, and timsTOF Ultra DIA-PASEF.
All platforms identified ∼4000 proteins in common, with 98%
proteome coverage of data-dependent acquisition (DDA) identifications
using data-independent (DIA) methods and 92% concordance between next-generation
systems. Orbitrap Astral and timsTOF Ultra quantified >225,000
peptides
and 13,000 proteins, representing ∼800% and ∼300% greater
depth than Exploris 480 DDA, respectively. Furthermore, new-generation
platforms cut recommended sample size by ∼66%. Enhanced proteome
depth improved subcellular compartment annotations from 30% (DDA)
to 66% (next-generation platforms) and reactome pathway coverage from
58% (DDA) to 90% (next-generation platforms). Differential expression
analysis identified up to four times more phenotype-associated proteins
in DIA data sets, enriched in mitochondrial, ribosomal, and extracellular
components, with up to 44 enriched pathways. Importantly, proteins
uniquely detected showed no functional annotation bias. These findings
demonstrate that DIA acquisition on multivendor next-generation platforms
provides superior proteome coverage and more complete systems biology
assessment without introducing bias, enabling enhanced understanding
of complex biological systems.