Xijing Hospital

Phosphoproteomics is of great significance for deciphering the regulatory mechanisms of cell signaling. In order to overcome the limitations of current sample preparation methods, it is crucial to develop novel techniques that offer improved cost-effectiveness, ease of use, and reproducibility, while also reducing the reliance on hazardous reagents. We have innovatively developed a fast and effective all-in-one single-tube phosphoproteomics (FEAS-phospho) workflow, integrating protein extraction, digestion and phosphopeptide enrichment into a single-tube operation. TFA was used to efficiently extract proteins from cell and tissue samples within 3 min, without additional homogenization or sonication. By adjusting the enzyme-to-protein mass ratio, protein digestion time was reduced to from 16 h to 15 min. Meanwhile, cumbersome steps such as peptide desalting, lyophilization, and re-dissolution after digestion were omitted, allowing in-situ direct phosphopeptide enrichment from the protein digest, which provided an efficient and reliable solution for high-throughput phosphorylation analysis. This workflow greatly simplified the cumbersome operations in classic phosphoproteomics analysis, and the entire process from protein extraction to phosphopeptide enrichment could be completed within 2.5 h. With a micro-flow LC-MS/MS system, 5087 phosphorylation sites, 6247 phosphopeptides, corresponding to 2295 phosphoproteins were identified from 200 μg of MCF-7 protein digest under data-dependent acquisition (DDA) mode with a 15 min gradient. While the data-independent acquisition (DIA) mode identified more than 8400 phosphopeptides with the same quantity and gradient. The FEAS-phospho was further validated by phosphoproteome analysis of five different mouse tissue. The FEAS-phospho can be easily integrated into the automated phosphoproteomics workflow, and is expected to have broad applications in phosphoproteomics analysis.

Noise-induced hearing loss can lead to degeneration of spiral ganglion neurons (SGN), resulting in impairment of auditory signalling. However, the specific mechanism is unclear. In our study, we investigated the function of mitochondrial activity following noise exposure. Findings revealed a reduction in ATP synthesis and alterations in the levels of mitochondrial electron transport chain complexes II, Ⅲ, IV and complex I subunit ND3 reduction by the seventh day post-noise exposure. These findings indicate that SGN mitochondrial dysfunction can impair SGN function. This is further demonstrated by the reduced expression of the mitochondrial marker protein Tom20 and apoptosis-inducing factor (AIF) showed inner membrane but not outer membrane resulted in changes in mitochondrial ETC complexes. And the findings of calpain 2 but not calpain 1 high expression was accompanied in AIF reduction, suggesting AIF has not yet caused SGN deaths. Calpain 2 regulated decreased expression of AIF may relieve the low function of mitochondrion in SGN after noise.

Auditory verbal hallucinations (AVH) are prominent positive symptoms of schizophrenia that frequently prove resistant to conventional antipsychotic treatments. Low‐frequency repetitive transcranial magnetic stimulation (rTMS) has emerged as a promising intervention for AVH; however, the underlying neurobiological mechanisms mediating its efficacy remain incompletely understood.

In this study, schizophrenia patients with AVH were randomly assigned to either an active stimulation group or a sham control group. The active stimulation group received 1 Hz rTMS targeting the left temporoparietal junction (TPJ), while the sham group underwent an identical procedure without actual stimulation. Structural MRI scans were conducted before and after treatment to evaluate changes in gray matter volume (GMV). Further analyses examined associations between GMV changes and both gene expression profiles and neurotransmitter receptor densities.

Active rTMS stimulation resulted in a significant reduction in AVH symptoms and was associated with increased GMV in specific cortical regions related to sensory and cognitive processing. These structural changes correlated with gene sets enriched in neuroplasticity‐related pathways, such as cell morphogenesis, chromatin remodeling, and vesicle cytoskeletal trafficking. Notable changes were also observed in neurotransmitter receptor densities, particularly for serotonin (5HT1a), dopamine (D1), and glutamate (mGluR5) receptors. Multiple linear regression analysis identified specific hub gene expressions, such as ANK1, and patterns of neurotransmitter density, especially mGluR5, as significant predictors of GMV changes following rTMS stimulation.

Low‐frequency rTMS induced GMV changes, coupled with alterations in gene expression and neurotransmitter receptor densities, contribute to symptom alleviation in schizophrenia patients with AVH. These findings support rTMS as a promising intervention for targeting the neurobiological substrates underlying AVH in schizophrenia.