Abstract
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.