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.