Data-Independent Acquisition Parallel Accumulation–Serial Fragmentation (diaPASEF) Analysis of the Separated Zebrafish Lens Improves Identifications

Ocular lens fiber cells degrade their organelles during
differentiation
to prevent light scattering. Organelle degradation occurs continuously
throughout an individual’s lifespan, creating a spatial gradient
of young cortical fiber cells in the lens periphery to older nuclear
fiber cells in the center of the lens. Therefore, separation of cortical
and nuclear regions enables examination of protein aging. Previously,
the human lens cortex and nucleus have been studied using data-independent
acquisition (DIA) proteomics, allowing for the identification of low-abundance
protein groups. In this study, we employed data-independent acquisition
parallel accumulation–serial fragmentation (diaPASEF) proteomics
on a timsTOF HT instrument to study the zebrafish lens proteome and
compared results to a standard DIA method employed on an Orbitrap
Exploris 480 mass spectrometer. Using the additional ion mobility
gas phase separation of diaPASEF, peptide and protein group identifications
increased by over 200% relative to an Orbitrap DIA method in the zebrafish
lens. With diaPASEF, we identified 13,721 and 11,996 unique peptides
in the cortex and nucleus of the zebrafish lens, respectively, which
correspond to 1,537 and 1,389 protein groups. Thus, separation of
the zebrafish lens into cortical and nuclear regions followed by diaPASEF
analysis produced the most comprehensive zebrafish lens proteomic
data set to date.