Methodological Study On Distinguishing Psychological Stress States Based On Hair Proteomic

Objectives Long-term and chronic psychological stress is the main cause of mental diseases such as depression,anxiety disorders and other socially prevalent and difficult-to-treat psychiatric disorders.Depression,with a high suicide rate,is difficult to treat,which brings a serious burden to people’s health and social development.The clinical diagnosis of depression still relies mainly on the doctors’ subjective judgment and the results of patients’ subjective psychological tests.There is no accurate and sensitive clinical diagnostic technology which lead to the difficulties in precision diagnosis.Therefore,there is an urgent need to develop new techniques and markers that can accurately reflect human stress.Psychological stress leads to phenotypic changes such as hair loss and poor hair quality,suggesting that the composition of hair and its physicochemical properties may have the potential to characterize the stressful state of the human body.However,due to the complex structure of hair proteins,there is currently a lack of a proteomics approach based on low hair input.Accordingly,this study intends to establish a method for efficiently extracting hair protein,further analyze the protein composition in hair,and explore whether hair proteomic changes can serve as an indicator of psychological stress,and whether hair protein composition can be used to assess human stress status.Methods This study systematically investigated the efficiency of hair protein extraction by various extraction methods such as glass homogenizer grinding,liquid nitrogen grinding,room temperature oscillation,and high temperature heating;on this basis,the influence of hair protein lysis buffer components on protein extraction was discussed.Combining the method of in-gel digestion and combined double-enzyme digestion to analyze the hair proteome,firstly use the damaged sample to verify the efficiency of the method,then use the human hair sample to explore,and finally explore the changes of the hair proteome in chronic restraint stress mice.Results This study further improved a stable and efficient technique for hair protein extraction called PLEE(PTM Lab for protein extraction from hair with high efficiency).Protein extraction and proteomics analysis were performed with healthy and bleached damaged hair samples based on the PLEE method,which resulted in the identification of 864-975 peptides from 155-158 proteins and was 13.4% and 21.4%higher than the identification of the Mase method(724-790 peptides from 134-142proteins),demonstrating the high efficiency of the PLEE method.On the other hand,the data analysis showed that the sequence coverage of key proteins of the hair proteome(KRTAP4-6 and KRTAP4-7)was 71.9%-79.5%,which was significantly higher than the that of 53.8%-60% in the published literature,further demonstrating the high efficiency and deep coverage properties of the PLEE method.By quantitative proteomic analysis of hair samples before and after stress,,which identified 1553-1820 peptides of 283-301 proteins,and was 49.5% higher than the identification reported in the existing literature.The results of clustering analysis and PCA analysis showed that samples from pre-and post-stress states could be clearly distinguished.The differentially expression analysis was conducted with a filter criteria of fold change>2or fold change<0.5,and P-value <0.05.The expression and sequence coverage of KRT16,KRT17,KRT16 B and KRT82 keratin proteins were up-regulated in hair samples after stress.KEGG enrichment analysis suggested that stress may be related to estrogen signaling pathway.To further validate the results and findings based on human samples,this study evaluated the ability of the hair proteome to characterize psychological stress status by constructing a mouse model of chronic restraint-induced depression.Two experimental groups were treated with two antidepressant drugs,ketamine and YL0919.Behavioral experiments such as forced swimming experiment proved that the depressed mouse model and antidepressant drug intervention model were successfully constructed.The changes in the hair proteome landscape of mice before and after depression,before and after drug intervention were explored which showed that the hair protein expression pattern of mice changed significantly after depression compared with the control group,besides,some differentially expressed proteins were also produced after the pharmacological intervention.The results validate that hair proteomics can be used for the differentiation of stress states and the tracking of stress levels.Conclusion This study developed a stable and efficient hair protein extraction method and further improved a stable process for hair proteomics analysis.The preliminary study of hair proteomic changes under stress has laid the foundation for better use of hair proteomics to characterize stress states.Through the study of the stressed human and chronic restraint causing depression mouse model,it is shown that the hair proteome has high sensitivity and accuracy in characterizing psychological stress.