Influence of Different Sample Preparation Approaches on Proteoform Identification by Top-Down Proteomics

Top-down proteomics (TDP) has made significant advances in the past, and a plethora of sample preparation workflows have been developed. Here, we systematically investigated the influence of different sample preparation steps on proteoform and protein identifications, including cell lysis, reduction and alkylation, proteoform enrichment, purification, and fractionation. We found that all steps in sample preparation influence the subset of proteoforms identified, e.g., their number, confidence, physicochemical properties, and artificially generated modifications. The various sample preparations resulted in complementary identifications, significantly increasing the depth of the analysis. Overall, 15,089 proteoforms from 2,780 protein accessions of human Caco-2 cells were identified, providing the most comprehensive dataset of this cell line up to now. The results from our study can serve as a guideline for designing and adapting TDP sample preparation strategies to particular research questions.