Frontal cortex - proteomics in a mouse model of chronic heart failure

Bottom-up proteomic analysis of frontal cortex was performed using a Thermo Scientific Q Exactive Plus Orbitrap Mass Spectrometer and TOP-10 Data Dependent Acquisition (DDA). Data were processed using Thermo Proteome Discoverer (version 2.3) in conjunction with SEQUEST using default settings. SEQUEST used a curated database consisting of FASTA sequences extracted from UniProt (Mus musculus taxon identifier 10090). Parameters were set as follows: MS1 tolerance of 10 ppm; MS2 mass tolerance of 0.02 Da for Orbitrap detection; enzyme specificity was set as trypsin with two missed cleavages allowed; carbamidomethylation of cysteine was set as a fixed modification; and oxidation of methionine was set as a variable modification. The minimum peptide length was set to six amino acids. Peptide-spectrum-matches (PSMs) and protein identifications were filtered at 1% false discovery rate (FDR) threshold. For protein quantification and comparative analysis, we used the peak integration feature of the Proteome Discoverer software. For each identified protein, the average ion intensity of the unique peptides was used for protein abundance. The sham group was the control group.本研究针对额叶皮层开展自下而上蛋白质组学分析（bottom-up proteomic analysis），所用仪器为赛默飞世尔科技（Thermo Scientific）Q Exactive Plus 轨道阱质谱仪（Orbitrap Mass Spectrometer），并采用TOP-10数据依赖性采集（Data Dependent Acquisition, DDA）模式。数据处理采用赛默飞Proteome Discoverer软件（版本2.3）结合SEQUEST工具，所有参数均采用默认配置。SEQUEST所使用的数据库为经人工整理的FASTA序列库，该序列提取自UniProt数据库，对应物种为小家鼠（Mus musculus），分类学标识符为10090。具体参数设置如下：MS1质谱质量容差设为10 ppm；轨道阱检测模式下的MS2质谱质量容差设为0.02 Da；酶切特异性设定为胰蛋白酶，允许最多2个漏切位点；半胱氨酸的氨基甲酰甲基化修饰设为固定修饰；甲硫氨酸的氧化修饰设为可变修饰；肽段最小长度设定为6个氨基酸残基。肽段-谱匹配（Peptide-spectrum-matches, PSMs）与蛋白质鉴定结果均以1%的假发现率（false discovery rate, FDR）作为过滤阈值。在蛋白质定量与比较分析环节，本研究借助Proteome Discoverer软件的峰积分功能完成计算；对于每一个鉴定得到的蛋白质，采用其特有肽段的平均离子强度作为其蛋白丰度的表征指标。本研究以假手术组（sham group）作为对照组。