Bioinformatics Analysis of Top-Down Mass Spectrometry Data

Accompanying dataset to the protocol published in Methods in Molecular Biology. Click on the PURL link below in the "External Files" section to download the dataset.Traditional bottom-up mass spectrometry-based proteomics relies on the use of an enzyme, often trypsin, to generate small peptides (typically < 25 amino acids long). In top-down proteomics, proteins remain intact and are directly measured within the mass spectrometer. This technique, while inherently simpler than bottom-up proteomics, generates data which must be processed and analyzed using software tools “purpose-built” for the job. In this chapter, we will show the analysis of proteins from deconvolution and deisotoping through analysis with ProSight Lite, a free, vendor agnostic tool for the analysis of top-down mass spectrometry data. We will illustrate with two examples of intact protein spectra and discuss the iterative use of the software to characterize proteoforms and to discover the sites of post-translational modifications.NIH/NIGMS P41GM10856