A chemical reporter facilitates the detection and identification of lysine HMGylation on histones† †Electronic supplementary information (ESI) available: Experimental methods and figures. See DOI: 10.1039/c8sc02483a

Chemical reporter, HMGAM-yne, facilitates the detection and identification of lysine HMGylation on histones.

. Metabolic labeling of MDH2 and CPS1 by HMGAM-yne.      Table S1. The estimated incorporation rates of HMGylated lysine in histones.

Note S2. MS/MS spectra of lysine HMGylated peptides in histones.
Note S3. 1 H-and 13 C-NMR spectra for all the compounds synthesized in this study

Supplementary References
Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2018 HMGAM-yne-labelled cell lysate were reacted with azide-biotin (100 M, 5 mM stock in DMSO), TCEP (1 mM, 50 mM stock in ddH2O freshly prepared), TBTA (100 M, 10 mM stock in DMSO) and CuSO4 · 5H2O (1 mM, 50 mM stock in ddH2O freshly prepared) for 1.5 h at room temperature. Proteins were precipitated by acetone (4 volume) at -20 ℃ for overnight. Precipitated proteins were centrifuged at 3500 g for 5 min at 4 ℃ and washed with ice-cold methanol twice. To capture the biotinylated proteins by streptavidin beads, the air-dried protein pellet was dissolved in 2 mL PBS (with 20 mM EDTA, 4% SDS and 10% Glycerol) and subsequently heated at 75 ℃ for 10 min, diluted by PBS to reduce SDS concentration down to 0.5%. Pre-washed streptavidin beads were then incubated with this protein solution at room temperature for 1.5 h on end-over-end rotator. After sequentially washed six times with PBS (with 0.2% SDS), 6M Urea (in PBS with 0.1% SDS), 250 mM NH4HCO3 (with 0.05% SDS), the enriched proteins were eluted by incubating with elution buffer (25 mM Na2S2O4, 250 mM NH4HCO3 and 0.05% SDS) for 1 h. The eluted proteins were then dried down with SpeedVac.

Enzymatic Reactions.
The enzymatic activities of human Sirtuins were measured by detecting the removal of HMG group from H3K9hmg peptide. 5 M of each Sirtuin protein was incubated with 500 M of peptide and 1 mM of nicotinamide adenine dinucleotide (NAD) in a reaction buffer containing 20 mM Tris-HCl buffer (pH 7.5) and 1 mM DTT at 37 °C for 2 h. The reactions were stopped by adding 1/3 reaction volume of 20% TFA and frozen in liquid N2 immediately. Samples were then analyzed by LC-MS with a Vydac 218TP C18 column (4.6 mm × 250 mm, 5 m, Grace Davison). Mobile phases used were 0.05% TFA in water (buffer A) and 0.05% TFA in ACN (buffer B). The flow rate for LC was 0.6 mL/min. The peptide mixtures were eluted by buffer A for 10 min, then 0-30% buffer B over 10 min.
MS started to record at 10 min for each injection.

Isothermal Titration Calorimetry Measurements.
Experiments were performed at 25 o C on a MicroCal iTC200 titration calorimeter

Immunofluorescence
HeLa cells grown on cover slips were metabolically labeled with DMSO or HMGAM-yne, fixed with 3.7% PFA, permeabilized with 0.1% Triton X-100 and then reacted with 20 μM azide-rhodamine, 1 mM TCEP, 100μM TBTA and 1 mM CuSO4 for 1 h. Cells were incubated with DAPI for 5min, washed trice with PBST (0.1% tween 20 in PBS) and then subjected to a Zeiss LSM 510 laser scanning confocal microscope.

Cellular Fractionation
Mitochondrial and nuclear extraction were performed as previously described [1] .

Histone Extraction
Acid-extraction method was used to isolate histones from HeLa S3 cells [2] .

Sample Preparation for Mass Spectrometry
In-solution tryptic digestion of histone samples was carried out based on previous described protocols [3] . Histone extracts were in-solution digested either without chemical propionylation, or chemically propionylated before or after in-solution trypsin digestion.
Histone proteins or peptides were treated with propionic anhydride twice to make sure fully labeling. The final resulting peptides were enriched and desalted with the StageTips. The eluted peptides from the StageTips were dried down by SpeedVac and then resuspended in 0.5% acetic acid for analysis by LC-MS/MS.

Mass Spectrometry
Mass spectrometry was performed on an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). First, peptide samples in 0.1% formic acid were pressure loaded onto a self- with the previous report [4] . spectroscopic data match with the previous report [5] . To a solution of compound 2 (800 mg, 2.31 mmol) dissolved in 5 mL dry THF, the supernatant of the above Grignard reagent was slowly added at 0 ℃ under Ar protection.
The mixture was stirred at 0 ℃ for 1 hour and gradually warmed up to room temperature for another 1 hour. The reaction was quenched with 20 mL saturated NH4Cl solution and the aqueous layer was extracted with 30 mL ethyl acetate for 3 times. The organic layers were pooled and washed with 30 mL brine, dried over anhydrous Na2SO4. After filtration and concentration, the crude product was purified by flash column chromatography to afford compound 3 (892 mg, 80%) as colorless oil. 1
The resulting aqueous mixture was extracted with 30 mL ethyl acetate for 3 times. The organic layers were combined and washed with 1 M HCl for 3 times and dried over anhydrous Na2SO4. After filtration and concentration, the resulting residue was subjected to flash chromatography (hexane:EA = 6:1) to afford compound 5 (4 g, 79%) as white solid.

Preparation of 3-hydroxy-3-(prop-2-yn-1-yl)pentanedinitrile (6).
To the solution of compound 5 (2 g, 4.57 mmol) dissolved in 5 mL anhydrous DMSO, NaCN (671 mg, 13.7 mmol) was added. The mixture was kept stirring at room temperature overnight. The reaction was quenched by 20 mL water and the aqueous layer was extracted with 20 mL ethyl acetate for 3 times. The combined organic layers were washed with 20 mL water for twice and dried over anhydrous Na2SO4. After filtration and concentration, the resulting residue was subjected to flash column (hexane:EA = 6:1) to afford compound 6 (480 mg, 71%) as yellow liquid. 1

Preparation of 3-hydroxy-3-(prop-2-yn-1-yl)pentanedioic acid (7).
To the solution of compound 6 (271 mg, 1.83 mmol) dissolved in 5 mL MeOH, 5 mL 35% (v/v) NaOH aqueous solution was added slowly at 0 ℃. The resulting solution was kept stirring at 60 ℃ overnight. The mixture was diluted with 20 mL ethyl acetate and the organic layer was discarded after partition. The aqueous layer was carefully acidified to pH = 1 at 0 ℃ with concentrated HCl. The aqueous solution was extracted with 30 mL ethyl acetate for 3 times and organic layers were pooled and dried over anhydrous Na2SO4. After filtration and concentration, the resulting residue was subjected to flash column chromatography (MeOH: dichloromethane   was added to the mixture and pH was adjusted to 2 with 1 M HCl at 0 ℃. The mixture was extracted with 30 mL dichloromethane for 3 times and the organic layer was pooled and dried over anhydrous Na2SO4. After filtration and concentration, the crude product was