Optimization of LC-MS/MS Data Independent Acquisition (DIA) for Plasma Biomarker Discovery

Abstract

Plasma is accessible to use and it is highly valuable for clinical diagnostics. However, the analysis of plasma protein biomarkers faces two major challenges. First, plasma includes a dynamic range of the proteome. Second, the presence of approximately 12 abundant proteins like albumin complicates the analysis of low-abundance proteins. As disease diagnosis primarily relies on low-abundance proteins as plasma biomarkers, overcoming these challenges becomes absolutely necessary. Quantitative proteomics utilizing LC-MS, focusing on the comparison between the conventional DataDependent Acquisition (DDA) method and the alternative Data-Independent Acquisition (DIA) method. While DDA provides rapid analysis, its stochastic nature leads to low reproducibility, especially in complex plasma samples. In contrast, DIA, utilizing MS2 spectra from precursor windows, demonstrates higher reproducibility, proving beneficial for the analysis of abundant protein. In this study, I optimized plasma protein digestion methods by comparing three digestion approaches: insolution digestion, S-Trap, and FASP. The FASP method emerged as the most suitable among the three. Moreover, to alleviate interference from high protein concentrations during plasma analysis, I prepared undepleted plasma and compared it to depleted plasma. Both depleted and undepleted plasma were subjected to analysis using DDA and DIA methods. The utilization of DIA revealed a higher number of identified proteins and peptides, with depleted samples exhibiting a greater protein count than undepleted samples. Furthermore, a noticeable reduction in the contribution of the top 12 abundant proteins was observed in the depleted samples. FDA biomarkers and cancer biomarkers were more prominently identified in the depleted samples, and DIA demonstrated superior efficacy compared to DDA in uncovering a greater number of biomarkers. As a result, this study emphasizes the critical role of depletion and DIA methods in increasing the depth and accuracy of plasma sample analysis.