Nature Methods Global profiling of dynamic protein palmitoylation Brent R Martin, Chu Wang, Alexander Adibekian, Sarah E Tully & Benjamin F Cravatt Supplementary Figure 1 Supplementary Figure 2 Supplementary Note HDFP enhances 17-ODYA labeling and prevents palmitoylation turnover in HEK293T cells. Representative MS1 signals of peptides extracted for 12 proteins that show HDFP-stabilized dynamic palmitoylation. Synthetic methods Note: Supplementary Tables 1 8 are available on the Nature Methods website.
Supplementary Figure 1 a 17-ODYA Pulse Rhodamine-azide Hours: 2 4 6 HDFP: - + - + - + b Coomassie Blue 2-4 + - + - 6 + c d Pulse-Chase Chase Hrs: 0 4 Pulse-Chase Chase Hrs: 0 4 HDFP: - - + HDFP: - - + 17-ODYA Rhodamine-N3 75 anti-flag FLAG-GFPHRAS 50 37 25 HDFP enhances 17-ODYA labeling and prevents palmitoylation turnover in HEK293T cells. (a) Cells treated with HDFP show a time-dependent increase in the extent of 17-ODYA labeling in HEK293T cells. (b) Increased 17-ODYA in the presence of HDFP is independent of protein abundance, as demonstrated by coomassie staining after in-gel fluorescence analysis. (c) HDFP prevents the palmitoylation turnover on specific proteins in HEK293T cells. Cells pulsed for 2 hours, then washed and placed in chase media with excess palmitic acid with DMSO or with HDFP for 4 hours. (d) Over-expressed wild-type FLAG-HRAS palmitoylation is partially stabilized by HDFP after a 17-ODYA pulse-chase. Fluorescence signal is referenced to total abundance by anti-flag western blotting.
Supplementary Figure 2 IPI00118569.1 Gna13 Guanine nucleotide binding protein alpha 13 R.APMAAQGMVETR.V; Mono.mass: 1260.595483 0 500000 1000000 1500000 2.25/1 0 100000 200000 300000 2.07/0.99 25 30 35 40 25 30 35 40 0 500000 1500000 3.28/1 0e+00 2e+05 4e+05 6e+05 8e+05 1e+06 2.91/1 25 30 35 40 30 35 40 45 IPI00416906.2 Gnas Isoform XLas 1 of Guanine nucleotide binding K.IEDYFPEFAR.Y; Mono.mass: 1285.597907 R.VLTSGIFETK.F; Mono.mass: 1093.601929 0 10000 30000 50000 4.76/1 0e+00 4e+05 8e+05 5.01/1 50 55 60 65 35 40 45 50 0 5000 10000 15000 20000 3.88/0.97 0e+00 4e+04 8e+04 50 55 60 65 60 Representative MS1 signals of peptides extracted for 12 proteins that show HDFP-stabilized dynamic palmitoylation. Red spectra represent light peptides and blue spectra represent heavy peptides. Representative peptides are shown from 15 proteins. The same peptide is shown for both experiments 1 and 2 when available. In each experiment, two groups of samples were prepared with pairs of heavy and light cells treated separately in reciprocal order to demonstrate the enrichment changes are caused by the treatment, and not by protein abundance changes in either light or heavy cells. Vertical green lines delineate the area used for integrating areas used for ratio calculation. Dotted horizontal lines display the signal baseline. Asterisks mark triggered MS2 used for sequence identification.
IPI00119939.1 Lat Linker for activation of T cells family member R.ELPVSYDSTSTESLYPR.S; Mono.mass: 1942.91601 0e+00 2e+05 4e+05 1.97/1 0 200000 600000 1000000 2.06/1 45 50 55 60 65 0e+00 2e+05 4e+05 6e+05 8e+05 2.34/0.99 0 500000 1500000 2500000 2.38/1 60 45 50 55 60 IPI00137706.1 Mpp1 55 kda erythrocyte membrane protein ENSMUSG00 K.TLVLIGASGVGR.S; Mono.mass: 1141.68191 0e+00 4e+04 8e+04 0 50000 100000 150000 4.98/0.98 45 50 55 60 65 0 20000 40000 60000 80000 17.36/0.99 0 20000 40000 60000 80000 9.95/0.98 45 50 55 60 65
IPI00124046.1 Mpp6 Isoform Beta of MAGUK p55 subfamily member 6 K.TLVLIGAQGVGR.R; Mono.mass: 1182.708459 0e+00 2e+05 4e+05 9.27/0.99 0 50000 150000 250000 5.78/1 35 45 50 55 60 65 0 20000 60000 100000 22.98/1 0e+00 4e+04 8e+04 8.47/1 45 50 55 60 65 IPI00330599.1 Mtdh Protein LYRIC ENSMUSG00000022255 IPI00330599 K.TLPPAISAEPSITLSK.G; Mono.mass: 1623.908343 0 20000 60000 100000 140000 3.63/1 0e+00 1e+05 2e+05 3e+05 4e+05 2.27/1 60 50 55 60 65 0 50000 150000 250000 3.68/1 0 50000 150000 250000 4.81/0.97 45 50 55 60 50 55 60 65 70
IPI00315334.5 Nras neuroblastoma ras oncogene ENSMUSG00000027852 K.LVVVGAGGVGK.S; Mono.mass: 954.586218 0e+00 2e+05 4e+05 6e+05 8e+05 2.58/1 25 30 35 40 45 0 500000 1000000 1500000 2.37/1 25 30 35 40 45 0 500000 1500000 2500000 3.58/1 0 500000 1000000 1500000 2.68/0.98 25 30 35 40 45 30 35 40 45 IPI00124282.1 Rala Ras related protein Ral A precursor ENSMUSG00 R.ADQWNVNYVETSAK.T; Mono.mass: 1623.752906 0 50000 100000 150000 3.89/1 0 20000 60000 100000 20/1 30 35 40 45 30 35 40 45 50 0e+00 2e+05 4e+05 6.77/0.99 0 20000 40000 60000 80000 5.26/1 30 35 40 45 50
IPI00121388.5 Rnasek Activated spleen cdna, RIKEN full length en K.RKEYMVR. ; Mono.mass: 980.522573 0e+00 4e+04 8e+04 3.79/0.99 20 25 30 35 0e+00 4e+04 8e+04 2.25/0.98 20 25 30 35 0 20000 60000 100000 140000 4.41/0.98 0 20000 60000 100000 3.08/0.98 15 20 25 30 15 20 25 30 IPI00323822.1 Rras2 Ras related protein R Ras2 precursor ENSMUSG R.QVTQEEGQQLAR.Q; Mono.mass: 1385.689911 0e+00 1e+05 2e+05 3e+05 2.32/1 0e+00 4e+05 8e+05 3.52/0.99 15 20 25 30 15 20 25 30 0e+00 1e+05 2e+05 3e+05 4e+05 4.17/1 0e+00 2e+05 4e+05 6e+05 8e+05 3.09/1 15 20 25 30 15 20 25 30 35
IPI00135388.2 Sft2d3 SFT2 domain containing 3 ENSMUSG00000044982 K.ASRPAAAEPLLGAK.A; Mono.mass: 1350.761952 0 200000 600000 1000000 7.42/0.98 0e+00 2e+05 4e+05 7.48/1 25 30 35 40 25 30 35 40 45 0e+00 2e+05 4e+05 6e+05 8e+05 20/1 0e+00 2e+05 4e+05 6.78/0.89 25 30 35 40 45 30 35 40 45 50 IPI00122015.1 0910001A06Rik Protein FAM49B ENSMUSG00000022378 IP R.INNVPAEGENEVNNELANR.M; Mono.mass: 2094.993032 0 50000 100000 150000 20/1 0 10000 30000 50000 7.73/0.99 30 35 40 45 30 35 40 45 0 50000 100000 150000 20/1 0 50000 100000 200000 20/1 35 40 45 50
Supplementary Note: Synthetic methods 1-Iodohexadecane (1). Triphenylphosphine (1.3 g, 4.9 mmol) and imidazole (337 mg, 4.9 mmol) were dissolved in CH 2 Cl 2 (27.0 ml) and iodine (1.25 g, 4.9 mmol) was gradually added. The mixture was stirred at room temperature for 20 min and then a solution of 1-hexadecanol (1.0 g, 4.1 mmol) in CH 2 Cl 2 (27.0 ml) was added dropwise and the reaction was stirred for 20 min at rt. The reaction was diluted with hexanes (100 ml), filtered and concentrated to afford a yellow solid. Purification of this solid by flash chromatography (100% hexanes) gave 2 (526 mg, 30%) as a white solid. R f 0.91 (20% EtOAc:hexanes). 1 H NMR (400 MHz, CDCl 3 ): = 3.13 (t, J = 6.9 Hz, 2H), 1.79 1.74 (m, 2H), 1.35 1.32 (m, 2H), 1.21 (bs, 24H), 0.84 (t, J = 6.5 Hz, 3H). HRMS: m/z: calcd for C 16 H 34 I: 353.1705; found 353.1706 [M + H] +. Diethyl hexadecylphosphonate (2). 1 (526 mg, 1.5 mmol) was dissolved in triethyl phosphite (450 L, 2.6 mmol) and heated to 155 C for 4 h. The reaction was then cooled to rt and concentrated to afford 2 as a colorless oil (316 mg, 59%). R f 0.1 (10% EtOAc:hexanes). 1 H NMR (400 MHz, CDCl 3 ): = 4.09 3.99 (m, 4H), 1.71 1.63 (m, 2H), 1.54 1.51 (m, 2H), 1.29 1.16 (m, 26H), 1.27 (t, J = 7.0 Hz, 6H), 0.84 (t, J = 6.5 Hz, 3H). HRMS: m/z: calcd for C 20 H 44 O 3 P: 363.3028; found 363.3030 [M + H] +.
Ethyl hexadecylfluorophosphonate (3). 2 (316 mg, 0.87 mmol) was dissolved in CH 2 Cl 2 (3.3 ml) and cooled to 0 C. To this was added trimethysilyl bromide (140 L, 1.1 mmol) and the reaction was stirred for 15 min at 0 C and then for 2 h at rt. The reaction was quenched with H 2 O, concentrated, and azeotroped with toluene (3x) to afford a yellow oil. This material was dissolved in CH 2 Cl 2 (2.6 ml) and cooled to 0 C and diethylaminosulfur trifluoride (345 L, 2.6 mmol) was added dropwise. The reaction was stirred at 0 C for 10 min and then at room temperature for 15 min. It was quenched with ice cold 0.5 N HCl and the organic layer removed, washed with H 2 O, dried over MgSO 4, filtered and concentrated to afford a colorless oil. Purification of this oil by flash chromatography (50% EtOAc:hexanes) gave 3 (186 mg, 64% over 2 steps) as a white solid. R f 0.8 (50% EtOAc:hexanes). 1 H NMR (400 MHz, CDCl 3 ): = 4.24 4.17 (m, 2H), 1.87 1.79 (m, 2H), 1.63 1.59 (m, 2H), 1.37 1.30 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H), 1.21 (bs, 24H), 0.84 (t, J = 6.5 Hz, 3H). HRMS: m/z: calcd for C 18 H 39 FO 2 P: 337.2672; found 337.2675 [M + H] +. Ethyl octadec-17-ynylfluorophosphonate. Diethyl octadec-17-ynylphosphonate was synthesized as previosuly described 15. Diethyl octadec-17-ynyl phosphonate (41 mg, 106 mmol) was dissolved in 0.5 ml CH 2 Cl 2, 0.1 ml oxalyl chloride were added and the reaction was stirred at room temperature overnight. The mixture was diluted with CH 2 Cl 2 and extracted 1x with 0.5 M HCl, and 2x with water. The organic phase was dried over sodium sulfate, dissolved in 0.5 ml
CH 2 Cl 2, and 0.1 ml DAST was added. The reaction was stirred for 20 minutes at -78 C, and for another 20 minutes at room temperature, then diluted with CH 2 Cl 2 and extracted with water. The organic layer was dried with sodium sulfate, concentrated under reduced pressure, and purified by flash chromatography (50% EtOAc:hexanes) to yield the final product (34 mg, 50%). 1 H NMR (400 MHz, CD 3 OD): = 4.26 (m, 2H), 2.18 (td, J = 2.6, 7.1 Hz, 2H), 1.94 (t, 2.6 Hz, 1H), 1.88 (m, 2H), 1.64 (m, 2H), 1.53 (m, 2H), 1.38 (m, 5H), 1.26 (bs, 22H). HRMS: m/z: calcd for C 20 H 39 FO 2 P: 361.2666; found 361.2721 [M + H] +.