Protein O-GlcNAcylation and hexokinase mitochondrial dissociation drive heart failure with preserved ejection fraction_Proteomics data

For proteomics with pulled down biotinylated samples, MS-grade trypsin (Promega, Madison WI) was added each sample at an enzyme-to-substrate ratio of 1:50 for overnight digestion at 37 ⁰C. Digestion was halted with the addition of 10% formic acid to a final concentration of 0.5%. The peptides were de-salted with C18 spin columns and completely dried prior to running on the mass-spectrometer. LC-MS analysis was performed on Vanquish Neo UHPLC (Thermo Fisher, VN-S10-A-01) coupled to Orbitrap Exploris 240 mass spectrometer (Thermo Fisher, BRE725535). Analytical separation was conducted using a UHPLC C18 column (Ion Opticks, AUR3-15075C18-CSI). For each run, 1ug equivalent of sample was injected. The flow rate was set at 0.2 µl/min. Elution of peptides from analytical separation column was performed using a 120 min gradient between buffer A (0.1% Formic acid and 99.9% Optima LC/MS grade water) and buffer B (80% Acetonitrile, 19.9% Optima LC/MS grade water, and 0.1% formic acid): 0 % B at the beginning, 8 % B at 1 min, 28 % B at 86 min, 50 % B at 106 min, 100 % B from 107 to 110 min, 0 % B from 111 to 120 min.

Electrospray ionization was performed using a Nanospray Flex Ion Source (Thermo Fisher, ES071) and positive static spray voltage was set at 2200 V. For full Scan range was set to 350-1600 m/z with RF lens: 60%, Orbitrap resolution: 120,000, Normalized AGC Target (%): 300, Maximum injection time (ms): 25, microscans: 1, and Intensity threshold: 5.0e3.

Data-dependent acquisition (DDA) by TopN was performed through fragmentation isolated precursor ions with charges between +2 to +5. The following parameters were used for DDA: Dynamic exclusion mode: exclusion duration (s) = 30, mass tolerance: 5 ppm (low) and 5 ppm (high). Data dependent mode: cycle time (s): 2, ddMS2 parameters isolation window (m/z) = 1.5, Normalized collisional energy = 30%, Orbitrap resolution 15,000, scan range mode: define first mass, first mass (m/z) 200, Normalized AGC target (%): 100, Maximum injection time (ms) = 50.

Proteins were identified from the MS raw files using the Mascot search engine (Matrix Science, London, UK. version 2.5.1). MS/MS spectra were searched against the SwissProt Human database. All searches included oxidized Methionine, deamidated asparagine and aspartic Acid, and acetylated

N-term as variable modifications. Two missed tryptic cleavages were allowed. 1% false discovery rate cutoff was applied at the peptide level. Only proteins with a minimum of two peptides above the cutoff were considered for further study. Identified peptides/protein were visualized by Scaffold software (version 5.0, Proteome Software Inc., Portland, OR)."

针对经亲和下拉富集的生物素化样品的蛋白质组学实验，向每份样品中加入质谱级胰蛋白酶（MS-grade trypsin，Promega公司，美国威斯康星州麦迪逊市），酶与底物比例为1:50，于37 ℃下过夜酶解。以添加10%甲酸至终浓度0.5%的方式终止酶解反应。所得肽段经C18固相萃取小柱脱盐后完全冻干，随后进行质谱上机分析。液相色谱-质谱（LC-MS）分析在Vanquish Neo超高效液相色谱（UHPLC，Thermo Fisher公司，型号VN-S10-A-01）联用Orbitrap Exploris 240质谱仪（Thermo Fisher公司，序列号BRE725535）上完成。色谱分离采用UHPLC C18色谱柱（Ion Opticks公司，型号AUR3-15075C18-CSI）。每次进样量为1 μg等效样品，流速设置为0.2 μl/min。肽段的洗脱采用120 min二元梯度流动相，流动相A为0.1%甲酸与99.9% Optima LC/MS级纯水，流动相B为80%乙腈、19.9% Optima LC/MS级纯水及0.1%甲酸：初始阶段为0% B，1 min时升至8% B，86 min时升至28% B，106 min时升至50% B，107~110 min维持100% B，111~120 min恢复至0% B。

电喷雾电离采用Nanospray Flex离子源（Thermo Fisher公司，型号ES071），正离子模式静态喷雾电压设置为2200 V。全扫描范围设为350~1600 m/z，射频透镜（RF lens）参数为60%，轨道阱分辨率为120000，归一化自动增益控制目标值（Normalized AGC Target, %）为300，最大注入时间为25 ms，微扫描次数为1，强度阈值为5.0×10³。

采用TopN模式的数据依赖性采集（Data-dependent acquisition, DDA）对电荷态为+2至+5的前体离子进行碎裂分离。DDA参数设置如下：动态排除模式：排除时长为30 s，质量容忍度为±5 ppm；数据依赖模式：循环时长为2 s，数据依赖二级质谱（ddMS2）参数：分离窗口为1.5 m/z，归一化碰撞能量为30%，轨道阱分辨率为15000，扫描范围模式设置为定义起始质量，起始质量为200 m/z，归一化自动增益控制目标值为100%，最大注入时间为50 ms。

采用Mascot搜索引擎（Matrix Science公司，英国伦敦，版本2.5.1）从质谱原始文件中鉴定蛋白质。将MS/MS谱图与SwissProt人类数据库进行比对。所有检索均将甲硫氨酸氧化、天冬酰胺与天冬氨酸脱酰胺以及N端乙酰化设为可变修饰，允许最多2处胰蛋白酶漏切位点。在肽段水平设置1%的假发现率（false discovery rate, FDR）筛选阈值，仅保留至少包含2条符合阈值要求的肽段的蛋白质用于后续分析。所鉴定的肽段与蛋白质通过Scaffold软件（版本5.0，Proteome Software Inc.公司，美国俄勒冈州波特兰市）进行可视化展示。