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.