We did not detect H3K4me3, yet our data suggest other combinations and modifications may be worth exploring. quantified proteoforms and determined Garenoxacin Mesylate hydrate changes in CD8 T cell histone PTMs over the course of infection. < 0.0001) with pairing evaluated by Spearman correlation (** < 0.0001). Inset: the number of enhancer PTMs (H4-K8ac, H3-K36me2, H3-K9ac, H2B-K12ac, H4-K5ac, H3-K36me3, H4-K12ac, H4-R3me2, H3-K36ac, H3-K27ac, H2A-S1p, H4-K16ac, H3-K23ac, H3-K18ac, and H2A-K5ac) were compared for the activated and na?ve with paired = 0.0046). To better compare histone proteoforms from na?ve and activated T cells, we normalized one representative dataset with the total Garenoxacin Mesylate hydrate histone proteoform intensities ABI2 within the na?ve and activated T cells (Table S2). H2A accounted for the largest percentage of histone Garenoxacin Mesylate hydrate cores in activated T cells. We found acetylation of serine 1 on H2A (S1ac) was increased in relative intensity (i.e., 23.4% versus 6.5%) and in the number of unique sequences identified in activated versus na?ve T cells (Tables S1 and S2). The relative abundance of proteoforms with phosphorylated serine 1 was also higher in activated versus na?ve T cells (i.e., 21.3% versus 7.0%) (Table S2). Overall, H2B showed very few differences in counts or relative abundance for na?ve and activated T cell subsets (Tables S1 and S2). However, both the fragment numbers and peak intensity of P1ac were higher for H2B from activated T cells (Figure 2b and Table S1). Interestingly, we identified Garenoxacin Mesylate hydrate four times more H2BS14p fragment ions from activated T cells than from na?ve (i.e., 24 to 6). The relative abundance of H3 was the same irrespective of activation (Table S2). H4 was the dominant species in the na?ve T cells. Acetylation of serine 1 accounted for 38.1% of the proteoforms detected in na?ve and only 13.6% in activated T cells. Indeed, when we compared acetylation of the first twenty amino acids on H4, it encompassed 50.7% of all species from na?ve T cells versus 20.5% from activated T cells. Apart from these exceptions, the majority of modified species we identified were present in histones from na?ve and activated T cells to a similar extent (Tables S1 and S2). To determine if individual modifications changed following activation, compared the total number of times each PTM was identified on the proteoforms of each core histone family member (Figure 2e). The distribution and median of PTMs per core histones from na?ve and activated T cells were significantly different (Figure 2e). Six modifications were detected in na?ve but not Garenoxacin Mesylate hydrate in activated T cells: H2BK108ac H3T3p, H3K4ac, H3R8me2, H3K9ac, and H4K8ac (Figure 2b,c and Table S1). Of these, H3K9ac and H4K8ac are associated with active gene expression, while H3R8me2 is associated with repression [53,54,55,56]. H3 lysine 27 was also acetylated; this activation signal was detected twice as many times for na?ve T cells than for activated (i.e., 108 versus 51 fragments corresponding to 6 versus 3 unique proteoforms). Overall, we detected significantly more known enhancers of gene expression for na?ve T cells (Figure 2e inset). There was not a significant difference in total repressive marks between na?ve and activated T cells (DNS). However, we found a reduction in H3K27me2 and H3K27me3 repressive marks in activated T cells (i.e., 102 versus 68 fragments) (Figure 2e). This was consistent with previous findings , and this trend also occurred for dimethylations and monomethylation of lysine 27 (Table S1 and File S1A,B). Given H3K27me3 is associated with repression of transcription of genes in loci related to effector function, these.