ZDHHC_metabolomics_chemgen_2023.zip

MetabolomicsAcyl- and probe-Coenzyme A analysis.HEK293T cells were seeded in 6-well plates, grown to 70% confluency in media containing 0.5% FBS and treated with 30 mM YnPal or 18-Bz for 2 h. Cells were dislodged into their growth media by pipetting and pelleted by centrifugation (500 x g, 5 min). The cell pellet was washed twice by resuspending in ice-cold PBS and pelleting by centrifugation.Sample extractionTo each sample, 400 µL chloroform was added and vortexed for ~1 min, followed by addition of 200 µL methanol and a repeated vortex. Samples were incubated in a water bath sonicator (4°C, 1 h), with 3 × 8 min sonication pulses, followed by centrifugation (4°C, 10 min, 17,000 x g). The supernatant was transferred to a new Eppendorf 1.5 mL tube (E1). The pellet was re-extracted with 450 µL methanol:water (2:1 v:v, containing internal standard, 13C3-Malonyl-CoA), sonicated (8 min, 4°C) and centrifuged, as above. The supernatant was added to the first extract (E1). Combined extracts were dried using a speedvac concentrator, re-suspended in 350 µL chloroform:methanol:water (1:3:3, v/v), and centrifuged, as above. The upper, aqueous phase containing the polar metabolites (including probe, probe-CoA, and acyl-CoA molecules) was dried using the speedvac concentrator and resuspended in 100 µL acetonitrile/ammonium carbonate 20 mM (7:3, v/v) for LC-MS injection.Liquid chromatography-mass spectrometry (LC-MS)Chromatography conditions:Chromatography prior to all mass spectrometry was performed using an adaptation of a method previously described61. Samples were injected into a Dionex UltiMate 3000 LC system (Thermo Fisher) with a Phenomenex Luna C18(2) 100 Å (100 x 2 mm, 3 μm) column coupled with a SecurityGuard C18 guard column (4 x 2 mm). Analytes were separated using 20 mM ammonium carbonate in water (Optima HPLC grade, Sigma Aldrich) as solvent A and acetonitrile (Optima HPLC grade, Sigma Aldrich) as solvent B at 0.3 mL/min flow rate. Elution began at 5% Solvent B, maintained for 3 min, increased to 100% B over 12 min, followed by a 3 min wash of 100% B and subsequent 3 min re-equilibration to 5% B. Other parameters were as follows: column temperature, 30°C; injection volume, 10 μL; needle wash, 50% methanol; autosampler temperature, 4°C.High resolution mass spectrometryPost-chromatography, high resolution (HR) MS was performed with positive and negative polarity switching using a Q-Exactive Orbitrap (Thermo Fisher) with a HESI-II (Heated electrospray ionization) probe. MS parameters were as follows: spray voltage, 3.5 kV and 3.2 kV (for positive and negative modes, respectively); probe temperature, 320°C; sheath and auxiliary gases, 30 and 5 arbitrary units (au), respectively; full scan range: 100 to 1300 m/z with settings of AGC target and resolution as Balanced and High (3 × 106 and 70,000), respectively. Data were recorded using Xcalibur 3.0.63 software (Thermo Fisher). Mass calibration was performed for both ESI polarities before analysis using the standard Thermo Fisher Calmix solution. Qualitative analysis was performed using Xcalibur FreeStyle 1.8 SP1 and Tracefinder 5.1 software (Thermo Fisher) according to the manufacturer’s workflows. Masses, retention times, and fragmentation of all relevant sample-derived molecules were compared to authentic chemical standards.MS/MS MS parameters were optimized by direct infusion of 16 μM acyl-CoAs dissolved in 10 mM MeOH/ammonium acetate at 5 μL/min into an TSQ Quantiva triple quadrupole MS (Thermo Fisher). The heated electrospray was set in positive mode with the following parameters: capillary voltage, 3472 V; sheath gas, 60 au; aux gas, 10 au; sweep gas, 1 au; ion transfer tube temp, 325°C; vaporizer temp, 275°C. A selected reaction monitoring (SRM) function was applied for the simultaneous detection of acyl-CoA and probe-CoA molecules with RF lens and collision energies as shown in the Supplementary Table 7. Data were recorded using the Xcalibur 4.0.27.10 software and analysed using QuanBrowser 4.5.445.18 (Thermo Fisher).

代谢组学酰基与探针辅酶A分析。将HEK293T细胞接种于6孔板中，在含有0.5%胎牛血清的培养基中培养至70%汇合度，随后用30 mM YnPal或18-Bz处理2小时。通过移液枪将细胞从培养基中释放并经离心（500 x g，5分钟）形成细胞沉淀。将细胞沉淀在冰冷的磷酸盐缓冲盐溶液（PBS）中悬浮两次，并再次离心。样品提取：向每个样品中加入400 µL氯仿，涡旋约1分钟，随后加入200 µL甲醇并重复涡旋。将样品在恒温水浴超声仪中孵育（4°C，1小时），使用3 × 8分钟的超声脉冲，随后离心（4°C，10分钟，17,000 x g）。将上清液转移至新的Eppendorf 1.5 mL管（E1）。将沉淀用450 µL甲醇:水（2:1 v:v，含有内标13C3-丙二酰辅酶A）重新提取，超声（8分钟，4°C）并离心，如上所述。将上清液加入第一次提取液（E1）。将合并的提取液用旋蒸浓缩仪浓缩干燥，用350 µL氯仿:甲醇:水（1:3:3，v/v）重新悬浮，并如上所述离心。使用旋蒸浓缩仪将含有极性代谢物（包括探针、探针辅酶A和酰基辅酶A分子）的上层水相干燥，并用100 µL乙腈/碳酸铵20 mM（7:3，v/v）重新悬浮，用于LC-MS注入。液相色谱-质谱联用（LC-MS）色谱条件：在所有质谱分析之前，采用61号文献中描述的方法的变体进行色谱分析。样品注入Dionex UltiMate 3000 LC系统（Thermo Fisher），配备Phenomenex Luna C18(2) 100 Å（100 x 2 mm，3 μm）柱和SecurityGuard C18保护柱（4 x 2 mm）。分析物使用20 mM碳酸铵水溶液（Optima HPLC grade，Sigma Aldrich）作为溶剂A和乙腈（Optima HPLC grade，Sigma Aldrich）作为溶剂B，在0.3 mL/min的流速下分离。洗脱从5%溶剂B开始，维持3分钟，逐渐增加到100% B，持续12分钟，随后用100% B清洗3分钟，并随后重新平衡至5% B。其他参数如下：柱温，30°C；注入体积，10 μL；针头清洗，50%甲醇；自动进样器温度，4°C。高分辨率质谱分析：色谱分离后，使用Q-Exactive Orbitrap（Thermo Fisher）和HESI-II（加热电喷雾电离）探头进行正负极性切换的高分辨率（HR）MS分析。MS参数如下：喷雾电压，3.5 kV和3.2 kV（分别对应正负模式）；探头温度，320°C；鞘气和辅助气，分别为30和5任意单位（au）；全扫描范围：100到1300 m/z，AGC目标和分辨率设置为平衡和高（3 × 106和70,000），分别。数据使用Xcalibur 3.0.63软件（Thermo Fisher）记录。在分析前使用标准Thermo Fisher Calmix溶液对ESI两种极性进行质量校准。使用Xcalibur FreeStyle 1.8 SP1和Tracefinder 5.1软件（Thermo Fisher）根据制造商的工作流程进行定性分析。将所有相关样品衍生物的质量、保留时间和裂解与标准化学物质进行比较。MS/MS MS参数通过将16 μM酰基辅酶A溶解在10 mM甲醇/乙酸铵中，以5 μL/min的速度直接注入TSQ Quantiva三重四极杆MS（Thermo Fisher）中进行了优化。加热电喷雾在正模式下设置，参数如下：毛细管电压，3472 V；鞘气，60 au；辅助气，10 au；扫描气，1 au；离子传输管温度，325°C；蒸发器温度，275°C。应用选择反应监测（SRM）功能，同时检测酰基辅酶A和探针辅酶A分子，RF透镜和碰撞能量如补充表7所示。数据使用Xcalibur 4.0.27.10软件记录，并使用QuanBrowser 4.5.445.18（Thermo Fisher）软件进行分析。