• Caroline J DeHart, Ryan T Fellers, Luca Fornelli, Neil L Kelleher, and Paul M Thomas.
• Departments of Chemistry, Molecular Biosciences, the Proteomics Center of Excellence and the Robert H. Lurie Comprehensive Cancer Center at the Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA.
• Methods Mol. Biol. 2017 Jan 1; 1558: 381-394.

AbstractTraditional 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 intact protein spectra through deconvolution, deisotoping, and searching 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 fragmentation spectra and discuss the iterative use of the software to characterize proteoforms and discover the sites of post-translational modifications.

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