Advancing serum peptidomic profiling by data-independent acquisition for clear-cell renal cell carcinoma detection and biomarker discovery

The analysis of serum endogenous peptides holds promise for disease study and drug discovery, whereas it is relatively unexplored given its challenges in analysis reproducibility and reliability. Here, we developed a streamlined detection platform for high-sensitive and reproducible serum peptidome profiling by data-independent acquisition (DIA) strategy. Compared to the classic data-dependent acquisition (DDA), our developed DIA approach can quantify almost twice the number of peptides with half the median coefficients of variation detected for DDA. The platform enables reproducible profiling of thousands of peptides and their post-translational modifications after simple sample preparation. The developed platform was subsequently utilized in the serum peptidome study of clear-cell renal cell carcinoma (ccRCC). A total of 31 ccRCC patients and 31 healthy volunteers were enrolled. Significant differences in serum peptidome patterns were observed between the two groups, allowing us to distinguish ccRCC patients from healthy volunteers clearly. A total of 833 modified peptides were found significantly changed in the ccRCC patients. The study demonstrated the high potential of serum peptidome in cancer detection and the feasibility and advantage of applying the DIA-based platform on large-scale serum peptidomic analysis for biomarker discovery. Significance: Serum peptidomic study proves to be challenging given its low abundance and instability of endogenous peptides. In this study, we developed a fast, reproducible and accurate detection platform by DIA-based MS method for streamlined serum peptidome profiling. The developed platform was then utilized in the serum peptidome study of ccRCC. To our knowledge, this is the first report to apply DIA strategy to disease related peptidomic studies. The large difference in serum peptidome profiles enabled us to distinguish ccRCC patients from healthy volunteers clearly, illuminating the great potential of serum peptidome in cancer diagnosis. The discovered significantly changed peptides provided a better understanding of the pathophysiological changes in ccRCC.