OBJECTIVE To establish a proteomic pre-processing protocol for low input samples (1×104 cells) in order to map the proteomic aging atlas of individual mouse antral follicles and elucidate the aging patterns of mouse antral follicles.METHODS Using cell lines and primary mouse cells,the protocol was developed by optimizing lysis buffers,enzymatic digestion,and ultrasonication for 1 × 104 cells (about 1 μg protein).The sample pre-processing protocol established in this study was evaluated by comparing it with two commercial sample preparation kits:iST kit,EasyPept-Ex (Ex kit),in terms of protein identification overlap,overall grand average of hydropathy (GRAVY),theoretical isoelectric points,and protein molecular weight.The sample suitability of the current sample pretreatment process was assessed using primary spleen and liver cell samples of mice at three different scales:100,1000,and 10000.Using this sample pre-processing approach in conjunction with the highly sensitive timsTOF Pro 2 mass spectrometer,proteome maps of individual antral follicles from young-aged (2 months),middle-aged (12 months),and old-aged (22 months) mice were generated.Differential and time-series analyses identified age-related proteins,and elucidated the aging patterns of mouse antral follicles.RESULTS A proteomic pre-processing protocol for 1 × 104 cell samples was established,requiring only one single-step operation for lysis,reduction,and alkylation,with enzymatic digestion not necessitating ultrasonication.We found that for cell samples on the order of 1 × 104,increasing the concentration of sodium deoxycholate in the lysis buffer from 1％ to 10％ enhanced the number of identified proteins from 4089 to 4389,demonstrating improved lysis efficiency (P＜0.05).The addition of ammonium bicar-bonate buffer (50 mmol·L-1,90 μL) to the lysis solution significantly increased the number of identified proteins from 2579 to 4389 (P＜0.01).Both single trypsin and mixed enzyme (trypsin/Lys-C) treatments yielded similar proteolytic outcomes,identifying approximately 3950 proteins each.Reducing the diges-tion time from overnight to 0.5 hours increased the number of identified proteins from 4299 to 4632 (P＜0.05),thus saving time while achieving higher protein identification yields.Compared to commercial kits,approximately 92.3％of the proteins identified by our protocol could also be identified by the iST kit or Ex kit.Our protocol demonstrated no significant bias in terms of hydrophobicity,theoretical isoelectric points or molecular weight,indicating robust performance.As the number of cells in the sample increased,the variety of identified proteins increased significantly.For instance,in samples containing 100,1000,and 10000 cells,525,1650,and 3210 proteins were identified in primary mouse spleen cells,respectively,compared with 366,1160,and 3590 proteins in primary mouse liver cells (P＜0.05).Finally,using this protocol,proteomic profiling of individual antral follicles from young,middle-aged,and old mice (three follicles per age group) was performed,with each follicle identifying over 7500 proteins on average.Based on this data,principal component analysis was conducted in this study,revealing significant differences in protein expression profiles of antral follicles at different age stages,confirming that age made a big difference to the physiological state of follicles.Additionally,we observed a signifi-cant downregulation of chromosome separation-associated proteins in aging mouse follicles (P＜0.01),suggesting potential disruptions in chromosome segregation and meiotic dysregulation.Further analysis revealed 739 proteins significantly correlated with age,among which 378 exhibited positive correlations and 361 negative correlations.CONCLUSION This study provides a low-cost,easy-to-operate,high-throughput,and highly sensitive proteomic sample pre-processing protocol for low input samples.It has constructed dynamic proteome maps of individual antral follicles at different ages in mice,offering high-quality data resources for basic research on follicular aging and providing new insights for exploring potential therapeutic targets for ovarian aging and the development of drugs to enhance fertility.

OBJECTIVE To establish a proteomic pre-processing protocol for low input samples (1×104 cells) in order to map the proteomic aging atlas of individual mouse antral follicles and elucidate the aging patterns of mouse antral follicles.METHODS Using cell lines and primary mouse cells,the protocol was developed by optimizing lysis buffers,enzymatic digestion,and ultrasonication for 1 × 104 cells (about 1 μg protein).The sample pre-processing protocol established in this study was evaluated by comparing it with two commercial sample preparation kits:iST kit,EasyPept-Ex (Ex kit),in terms of protein identification overlap,overall grand average of hydropathy (GRAVY),theoretical isoelectric points,and protein molecular weight.The sample suitability of the current sample pretreatment process was assessed using primary spleen and liver cell samples of mice at three different scales:100,1000,and 10000.Using this sample pre-processing approach in conjunction with the highly sensitive timsTOF Pro 2 mass spectrometer,proteome maps of individual antral follicles from young-aged (2 months),middle-aged (12 months),and old-aged (22 months) mice were generated.Differential and time-series analyses identified age-related proteins,and elucidated the aging patterns of mouse antral follicles.RESULTS A proteomic pre-processing protocol for 1 × 104 cell samples was established,requiring only one single-step operation for lysis,reduction,and alkylation,with enzymatic digestion not necessitating ultrasonication.We found that for cell samples on the order of 1 × 104,increasing the concentration of sodium deoxycholate in the lysis buffer from 1％ to 10％ enhanced the number of identified proteins from 4089 to 4389,demonstrating improved lysis efficiency (P＜0.05).The addition of ammonium bicar-bonate buffer (50 mmol·L-1,90 μL) to the lysis solution significantly increased the number of identified proteins from 2579 to 4389 (P＜0.01).Both single trypsin and mixed enzyme (trypsin/Lys-C) treatments yielded similar proteolytic outcomes,identifying approximately 3950 proteins each.Reducing the diges-tion time from overnight to 0.5 hours increased the number of identified proteins from 4299 to 4632 (P＜0.05),thus saving time while achieving higher protein identification yields.Compared to commercial kits,approximately 92.3％of the proteins identified by our protocol could also be identified by the iST kit or Ex kit.Our protocol demonstrated no significant bias in terms of hydrophobicity,theoretical isoelectric points or molecular weight,indicating robust performance.As the number of cells in the sample increased,the variety of identified proteins increased significantly.For instance,in samples containing 100,1000,and 10000 cells,525,1650,and 3210 proteins were identified in primary mouse spleen cells,respectively,compared with 366,1160,and 3590 proteins in primary mouse liver cells (P＜0.05).Finally,using this protocol,proteomic profiling of individual antral follicles from young,middle-aged,and old mice (three follicles per age group) was performed,with each follicle identifying over 7500 proteins on average.Based on this data,principal component analysis was conducted in this study,revealing significant differences in protein expression profiles of antral follicles at different age stages,confirming that age made a big difference to the physiological state of follicles.Additionally,we observed a signifi-cant downregulation of chromosome separation-associated proteins in aging mouse follicles (P＜0.01),suggesting potential disruptions in chromosome segregation and meiotic dysregulation.Further analysis revealed 739 proteins significantly correlated with age,among which 378 exhibited positive correlations and 361 negative correlations.CONCLUSION This study provides a low-cost,easy-to-operate,high-throughput,and highly sensitive proteomic sample pre-processing protocol for low input samples.It has constructed dynamic proteome maps of individual antral follicles at different ages in mice,offering high-quality data resources for basic research on follicular aging and providing new insights for exploring potential therapeutic targets for ovarian aging and the development of drugs to enhance fertility.