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Summary Data-independent acquisition mass spectrometry (DIA-MS) is a powerful technique that enables relatively deep proteomic profiling with superior quantification reproducibility. DIA data mining predominantly relies on a spectral library of sufficient proteome coverage that, in most cases, is built on data-dependent acquisition-based analysis of the same sample. To expand the proteome coverage for a pre-determined protein family, we report herein on the construction of a hybrid spectral library that supplements a DIA experiment-derived library with a protein family-targeted virtual library predicted by deep learning. Leveraging this DIA hybrid library substantially deepens the coverage of three transmembrane protein families (G protein-coupled receptors, ion channels, and transporters) in mouse brain tissues with increases in protein identification of 37%–87% and peptide identification of 58%–161%. Moreover, of the 412 novel GPCR peptides exclusively identified with the DIA hybrid library strategy, 53.6% were validated as present in mouse brain tissues based on orthogonal experimental measurement., Graphical Abstract, Highlights • A virtual library is built for a selected protein family using deep learning models • The hybrid library strategy vastly deepens the coverage for the targeted protein family • About 53.6% of novel GPCR peptides identified with the DIA hybrid library are validated • Extend the strategy to deep mapping of multiple transmembrane protein families, Analytical Chemistry; Biological Sciences; Classification of Proteins; Proteomics

Summary The axon initial segment (AIS) cytoskeleton undergoes rapid and irreversible disruption prior to cell death after injury, and loss of AIS integrity can produce profound neurological effects on the nervous system. Here we described a previously unrecognized mechanism for ischemia-induced alterations in AIS integrity. We show that in hippocampal CA1 pyramidal neurons Nav1.6 mostly preserves at the AIS after disruption of the cytoskeleton in a mouse model of middle cerebral artery occlusion. Genetic removal of neurofascin-186 leads to rapid disruption of Nav1.6 following injury, indicating that neurofascin is required for Nav1.6 maintenance at the AIS after cytoskeleton collapse. Importantly, calcineurin inhibition with FK506 fully protects AIS integrity and sufficiently prevents impairments of spatial learning and memory from injury. This study provides evidence that calcineurin activation is primarily involved in initiating disassembly of the AIS cytoskeleton and that maintaining AIS integrity is crucial for therapeutic strategies to facilitate recovery from injury., Graphical Abstract, Highlights • Ion channels are mostly retained at the AIS after ischemic injury • Neurofascin is required for clustering ion channels at the AIS after ischemia • Calcineurin inhibition protects AIS structural integrity and function from ischemia • Calcineurin inhibition protects cognitive function against impairment by ischemia, Neuroscience; Molecular Neuroscience; Cellular Neuroscience