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 tissue from a bronchopulmonary dysplasia model (n=12), employing four acquisition strategies: Exploris 480 DDA/DIA, Astral HR-DIA, and timsTOF Ultra DIA-PASEF.