ECD- electron capture dissociation used on whole proteins ETD- electron transfer dissociation. Can analyze 50 resides. -showed that bivalent H3K27me1,2 and H3K4Me1 were on same peptide. -analyze fraction w. 1 Ac and one me and then see where they map using MS/MS you know they're on the same tail.

In vivo stable isotope labelling to quantify the modification levelsat specific residues in histones. -modify the purified protein in vitro w. deuterated acetic anhydride which makes all lysines acetylated in vivo have 42 Da acetyl group whereas nonAc lysines now have dueterated Ac 45 Da. This allows you to determine the ratio of deuterated to non-deuterated Ac at each K residue by MS/MS. SILAC- selective isotope labeling with aa in cell culture. -grow cells in normal vs. deuterated K media. Each group represents a condition to be compared. After growth mix equal number of cells from each culture and isolate histones. Combining cells prior to isolation protects agains error due to extraction, purification, and analysis. Calculate to ration of labeled to unlabeled from doublets that arise from stable isotope incorporation. Calculate ratio of labeles to unlabeled residue.

-KO all jmj proteins in yeast and monitored methylation levels in bulk by MS/MS. (only looked at H3!) V high resolution so they can differentiate the mass of trimethyl from acetyl. (LTQ-FTICR mass spectrometer). -quantified by comparing the percent of each peak in a given spectrum in mutant and wt. Therefore calculated percetn change. -only ecm5KO showed no effect. -H3K4me3 and H3K36Me3 went up in rph1 and jdh2KO strains respectively. -H3K36me and me2 went up in gis1 and jdh1 strains. -o/e Jmj proteins shows the inverse effect on the modificaitons. -in vitro assays show Rph1 acts on H3K36me3 and me2. -were able to purify GST-tagged jmjC domains in bacteria. -purified His tagged full length proteins from yeast (b/c full length proteins had solubility problems in bacteria. -only truncated Rph1 shows HDM activity in vitro. **need 20 ug of protein for in vitro assays -> ask josh - knocking out any of teh jmj protein had no phenotypic effect (only tried UV and 6AU) -rph1 growth defect was only observed on minimal media. (SD-Ura+Gal) -o/e Rph1 confers UV sensitivity. Catalytic activity is necessary for the sensitivity. -Rph1 represses phr1 which encoded a photolyase in response to UV damage. -o/e of rph1 lead to UV sensitivity and is dependent on demethylase activity. didn't test unmodifiable mutant -> stupid. -prev studies shows set2KO leads to 6Au resistance. o/e of rph1 and jdh1 show slight sensitivity to 6AU.-> consistent w. set2ko. Shows decreased levels of methylation decrease the amount of transcription taking place. therefore cells are less sensitivie to 6AU. -Jdh2 interacts w. Sas10 (o/e lead to derepression of MAT loci. Fun30 putative ATP helicase snf2 homologue. -suggests jdh2 may be part of a modifying/remodeling complex.