Top Down Proteomics of Laser Capture Microdissected Samples for the Cancer Moonshot

Label-free quantitative top-down proteomics of APOLLO tissue samples will be performed using the top-down proteomic pipeline that has been developed in the National Research Resource for Top Down Proteomics. We will quantify between 750-1,500 proteoforms from APOLLO cancer tissue samples. Proteins will be extracted from tumor tissue (~50 μg of protein required) and the 0-30 kDa proteome is separated into a single fraction using GELFrEE. Protein samples will then be separated by reverse-phase liquid chromatography and infused into an Orbitrap mass spectrometer. The resulting data will be searched using the platform above and precursor ionbased quantitation will be performed. Once these precursor ion areas are extracted, the data will be standardized and normalized before standard analysis of variance (ANOVA) is performed on a hierarchical linear model for each proteoform.