pyMS-Vis, an Open-Source Python Application for Visualizing and Investigating Deconvoluted Top-Down Mass Spectrometric Experiments: A Histone Proteoform Case Study.

We report the development of an open-source Python application that provides quantitative and qualitative information from deconvoluted liquid-chromatography top-down mass spectrometry (LC-TDMS) data sets. This simple-to-use program allows users to search masses-of-interest across multiple LC-TDMS runs and provides visualization of their ion intensities and elution characteristics while quantifying their abundances relative to one another. Focusing on proteoform-rich histone proteins from the green microalga Chlamydomonas reinhardtii, we were able to quantify proteoform abundances across different growth conditions and replicates in minutes instead of hours typically needed for manual spreadsheet-based analysis. This resulted in extending previously published qualitive observations on Chlamydomonas histone proteoforms into quantitative ones, leading to an exciting new discovery on alpha-amino termini processing exclusive to histone H2A family members. Lastly, the script was intentionally developed with readability and customizability in mind so that fellow mass spectrometrists can modify the code to suit their lab-specific needs.

An Improved Top-Down Mass Spectrometry Characterization of Chlamydomonas reinhardtii Histones and Their Post-translational Modifications.

We present an updated analysis of the linker and core histone proteins and their proteoforms in the green microalga Chlamydomonas reinhardtii by top-down mass spectrometry (TDMS). The combination of high-resolution liquid chromatographic separation, robust fragmentation, high mass spectral resolution, the application of a custom search algorithm, and extensive manual analysis enabled the characterization of 86 proteoforms across all four core histones H2A, H2B, H3, and H4 and the linker histone H1. All canonical H2A paralogs, which vary in their C-termini, were identified, along with the previously unreported noncanonical variant H2A.Z that had high levels of acetylation and C-terminal truncations. Similarly, a majority of the canonical H2B paralogs were identified, along with a smaller noncanonical variant, H2B.v1, that was highly acetylated. Histone H4 exhibited a novel acetylation profile that differs significantly from that found in other organisms. A majority of H3 was monomethylated at K4 with low levels of co-occuring acetylation, while a small fraction of H3 was trimethylated at K4 with high levels of co-occuring acetylation.