Biology subjects

Tomioka, R.

Publications and source records attributed to Tomioka, R..

3 recordsLinked to original sources

bioRxiv2025

Anatomical and Neurochemical Profiles of GABAergic Projection Neurons in the Mouse Inferior Colliculus

The inferior colliculus (IC) is a critical hub for the integration of auditory signals in the midbrain. Although both glutamatergic and GABAergic neurons in the IC project to the medial geniculate body (MGB), the detailed neurocircuitry and neurochemical properties of GABAergic projection neurons remain poorly understood. In this study, glutamate decarboxylase 67 (GAD67)-Cre mice and the viral vector were utilized to selectively visualize the axonal projections of GABAergic neurons in the IC. These neurons project predominantly to the medial division of MGB (MGv) and contralateral IC and minorly to the ventral nucleus of the trapezoid body. Importantly, GABAergic projections from both lemniscal and non-lemniscal regions of the IC primarily targeted the lemniscal division of the MGB, whereas those projections from the external cortex of the IC--part of the non-lemniscal pathway--additionally extended into non- lemniscal MGB subregions. Using a retrograde tracer, a substantial proportion of GABAergic projection neurons targeting the MGB and contralateral IC were positive for several neurochemical markers, implying that some GABAergic neurons send axon collaterals to both targets. Notably, GABAergic neurons constituted 10% of the total neuronal population in the IC, whereas GABAergic neurons accounted for approximately 20% of IC neurons projecting to the MGB or contralateral IC. Our results suggest that GABAergic projection neurons are more involved in the IC-MGB pathway than would be expected based on their proportion in the IC and may exert widespread influence on auditory processing via direct inhibition of the MGv and indirect modulation through suppression of the contralateral IC.

neuroscience↗

bioRxiv2025

Bottom-up proteomics under acidic conditions using protease type XIII from Aspergillus saitoi

Bottom-up proteomics is a powerful technique for comprehensive analysis of proteins by proteolytic cleavage followed by liquid chromatography/tandem mass spectrometry to identify the resulting peptides. Trypsin is the gold-standard protease for bottom-up proteomics, though its cleavage specificity limits peptide identification, depending on the protein sequence. In addition, its optimal pH is weakly alkaline, which can cause modification artifacts such as deamidation. We hypothesized that these limitations might be overcome by using protease type XIII (P13ase) from Aspergillus saitoi, which is active at low pH. P13ase has been used for protein structural analysis by hydrogen-deuterium exchange mass spectrometry, but its cleavage preferences have not been clarified. Here, we show that P13ase primarily cleaves the C-terminal side of Lys, Arg, and Leu, and the optimal P13ase digestion conditions for bottom-up proteomics are pH 3.5, 37{degrees}C for 60 min. Under these conditions, sequence coverage of more than 90% was achieved for several proteins in HeLa cell extracts, which is unachievable with trypsin. In addition, P13ase digestion reduced artifacts such as deamidation products generated by cyclization reactions and subsequent hydrolysis. These results indicate that P13ase is a promising new tool for precision proteomics.

biochemistry↗

bioRxiv2023

One thousand samples per day capillary-flow LC/MS/MS for high-speed, high-sensitivity and in-depth proteomics

We developed a capillary-flow LC/MS/MS system with ultrahigh speed, enabling a throughput of 1,000 samples per day while maintaining high sensitivity and depth of analysis. In targeted LC/MS mode, 36 endogenous phosphopeptides in HeLa cells, including EphA2- derived phosphopeptide isomers, were successfully quantified with high selectivity and linearity by combining ion mobility separation. When 500 ng of HeLa cell digest was measured 100 times repeatedly in data-dependent acquisition mode, the coefficient of variation of retention time, peak intensity and number of identified peptides were on average 3.4%, 19.8%, and 6.0%, respectively. In data-independent acquisition mode, this system achieved the identification and quantification of 3,139 protein groups from a 100 ng HeLa cell digest and 2,145 protein groups from a sample of only 10 ng. The coefficient of variation of protein commonly quantified in the triplicate analysis ranged from 12 to 24% for HeLa digest samples ranging from 10 to 1000 ng. Finally, we applied this high-speed system to the spatial proteomics of the mouse brain, and succeeded in capturing the proteome distribution along a 96-sectioned brain structure in 135 minutes. This is the first LC/MS/MS system to achieve both more than 500 samples per day and more than 3000 identified protein groups ID with less than 100 ng human cultured cells simultaneously.

systems biology↗