ESPRESSO: a universal proteomic workflow based on a novel non-toxic dewaxing agent for FFPE samples

Introduction
FFPE tissue samples are invaluable resources for proteomics-based clinical and biomedical research. Formalin fixation and paraffin-embedding allow proteins to be stable for decades but make protein extraction and analysis more challenging than fresh frozen tissues. Traditionally, paraffin is removed by stepwise incubating samples with xylene, ethanol, several aqueous solutions with decreasing concentrations, and finally with water. Besides being time-consuming, researchers are unavoidably exposed to the hazards of xylene. Here, we presented a non-toxic, efficient, and cheap substitution of xylene which passed the careful evaluation of proteomic analysis. The streamlined proteomic method based on this substitution proves the feasibility of analyzing large tissue cohorts in a robust, timely, and unbiased manner.
Methods
Based on this, a non-toxic green dewaxing reagent that can completely replace xylene was screened, and a complete method system (ESPRTSSO) in DIA mode was established and evaluated. Subsequently, completely symmetrical FFPE and OCT samples were prepared based on mouse kidney tissues, and the biological differences between FFPE samples and OCT samples were systematically evaluated using different DIA library search modes of different protein search software (MaxDIA, DIA-NN, and Spectronaut), as well as comparing the effects of different libraries on protein quantification.
Results
The entire method reduces the processing time by approximately 4/5 compared to the traditional method, with the preparation process completed within 4 hours. The sensitivity of the method enables protein-level identification with a starting amount of 1µg protein. Furthermore, we tested the stability in human tumor FFPE samples, and the novel dewaxing agent can be adapted to other method systems, such as PCT. A systematic comparison of perfectly symmetrical FFPE and OCT samples from mice revealed that the difference between FFPE and OCT samples is much smaller than that of the biological samples. Furthermore, the construction of a hybrid library based on peptides from FFPE samples and peptides from OCT samples demonstrated that proteins lost during the preparation of FFPE samples can be retrieved. This improvement in peptide coverage per protein group enhances the overall reliability of the method.
Conclusion
In conclusion, we've developed a mass spectrometry pre-processing procedure for FFPE samples using a new non-toxic green dewaxing agent and conducted a thorough evaluation of the method in DIA mode.