Systematic comparison of the effects of various fixatives on the stability of the cellular proteome

Proteomics research represents a critical area within modern biomedical science. Fixatives, as essential components in proteomic analysis, play a pivotal role in maintaining proteome stability and analytical accuracy, while also exerting a substantial influence on experimental outcomes. However, there remains a limited understanding of how various fixatives affect proteome stability. In this study, we conducted a systematic comparison of commonly used fixation agents-specifically formaldehyde, paraformaldehyde, methanol, and ethanol-to evaluate how they affect the stability of the cellular proteome. Our results show that different fixatives lead to notably different levels of proteome stability. Importantly, formaldehyde solution and paraformaldehyde produce cross-linking effects that can significantly reduce protein solubility and hinder the efficiency of enzymatic digestion. Additionally, we analyzed the proteomic profiles of both drug-treated and untreated samples under different fixation conditions to better understand how these conditions may affect the quality and interpretation of mass spectrometry data. These results provide a scientific basis for selecting the most appropriate fixative in proteomics research. Our study also contributes to improving the compatibility between fixatives and mass spectrometry technologies, offering dependable technical support for biological and biomedical research. SIGNIFICANCE: We conducted a systematic comparison of commonly used fixation agents-specifically formaldehyde, paraformaldehyde, methanol, and ethanol-to evaluate how they affect the stability of the cellular proteome. These results provide a scientific basis for selecting the most appropriate fixative in proteomics research. Our study also contributes to improving the compatibility between fixatives and mass spectrometry technologies, offering dependable technical support for biological and biomedical research.