A Comprehensive Comparison of Sample Preparation and Normalization Methods for Bottom-Up Proteomics of Extracellular Vesicles

In this scientific study, we evaluated sample preparation methods for exosome proteomics along with data normalization techniques. We commenced by extracting exosomal proteins using three different lysis buffers: 8M urea, 1% sodium deoxycholate (SDC), and 0.1% sodium dodecyl sulfate (SDS). Through comparative analysis, we determined that 1% SDC was the most efficient lysis buffer, yielding the highest quality and quantity of protein extracts. Subsequently, we examined various data normalization methods to ensure reliable and reproducible results in proteomic profiling. Our findings highlight the critical role of optimal sample preparation and data normalization in exosome proteomics, which are essential for accurate biomarker discovery and understanding exosomal functions in biological processes. This research provides a foundation for standardized protocols in exosome proteomics and contributes to the enhancement of data quality in this burgeoning field.