Discovery of Unknown Posttranslational Modifications by Top-Down Mass Spectrometry

Protein encoding genes can undergo modifications posttranscriptionally and posttranslationally, yielding many different "proteoforms." The chemical diversity of such modifications is known to be important biomarkers of function within biological systems but is not completely understood. Top-down mass spectrometry is a valuable tool for the characterization of proteoforms, especially for histones that have complex combinations of posttranslational modifications (PTMs). In this chapter, we present a top-down liquid chromatography-mass spectrometry experimental and data analysis workflow for the identification of novel, unexpected modifications on histones. Proteoforms of interest are first discovered using the "open" modification search in TopPIC. Then target proteoforms are manually confirmed using the data visualization tool-LcMsSpectator, part of the Informed-Proteomics package. The workflow can be very helpful in targeted PTM analysis and can be expanded to other types of proteins for discovery of unknown PTMs.