Normal liver tissue proteome analysis

Liver tissue was homogenized and sonicated in the lysis buffer. Whole cell protein extract was reduced and alkylated and digested by trypsin. <br>Peptide mixture was analyzed on Q Exactive HFX mass spectrometer coupled to UHPLC chromatography system. Two dimensional fractionation of peptides included one more separation on Agilent 1200 Series RP chromatography in alkaline conditions with fractions collected and also analyzed by MS. The fractions were also analyzed by SRM SIS analysis to detect proteins encoded by the 18th human chromosome. The analysis was carried out at 6495 Triple Quad LC/MS system (Agilent). Stable isotope labelled peptides were used as internal standard.The Shotgun data was processed with MaxQuant software v1.6.3.4 by target/decoy method. Cleavage by trypsin, max number of missed cleavages - 2. Oxidation of M and Acetylation of protein N-term were set as variable modifications. Carbamidomethylaion was set as fixed modification.

将肝组织置于裂解缓冲液中进行匀浆并超声破碎。收集全细胞蛋白提取物，经还原、烷基化处理后用胰蛋白酶酶解。
肽混合物在与超高效液相色谱（UHPLC）联用的Q Exactive HFX质谱仪（Q Exactive HFX mass spectrometer）上完成分析。肽段的二维分级分离还包含在碱性条件下通过安捷伦1200系列反相液相色谱（Agilent 1200 Series RP chromatography）进行的额外分离步骤，收集分级组分后同样通过质谱（MS）进行分析。上述分级组分还通过选择反应监测-稳定同位素标准（SRM SIS）分析，以检测人类第18号染色体编码的蛋白质。该分析采用安捷伦6495三重四极杆液相色谱-质谱联用系统（6495 Triple Quad LC/MS system，Agilent）开展。实验以稳定同位素标记肽段作为内标。鸟枪法蛋白质组学（Shotgun proteomics）数据通过目标-诱饵（target/decoy）方法，使用MaxQuant软件v1.6.3.4进行处理。胰蛋白酶酶切，最大漏切位点数设为2。将甲硫氨酸（M）氧化及蛋白质N端乙酰化设为可变修饰。将半胱氨酸甲酰胺甲基化设为固定修饰。