Zhan Xianquan, Huang Yuda, Long Ying
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University; Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University; State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University; The State Key Laboratory of Medical Genetics, Central South University;
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University; Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University; State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University.
J Vis Exp. 2018 Apr 2(134):56739. doi: 10.3791/56739.
Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.
人类垂体腺瘤（PA）是一种常见肿瘤，发生于下丘脑 - 垂体 - 靶器官轴系统中的人体垂体，可分为临床功能性或非功能性PA（FPA和NFPA）。与FPA相比，NFPA由于血液中激素几乎不升高，早期诊断和治疗较为困难。我们的长期目标是使用蛋白质组学方法发现可靠的生物标志物，以阐明PA的分子机制，并识别有效的诊断、预后标志物和治疗靶点。本文介绍了有效的二维凝胶电泳（2DE）与质谱（MS）联用方法，用于分析人类PA蛋白质组，包括样品制备、二维凝胶电泳、蛋白质可视化、图像分析、胶内胰蛋白酶消化、肽质量指纹图谱（PMF）和串联质谱（MS/MS）。二维凝胶电泳 - 基质辅助激光解吸/电离质谱PMF（2DE - MALDI MS PMF）、2DE - MALDI MS/MS和2DE - 液相色谱（LC）MS/MS程序已成功应用于NFPA蛋白质组分析。使用高灵敏度质谱仪，在复杂PA组织分析中，通过2DE - LC - MS/MS方法在每个二维凝胶斑点中鉴定出许多蛋白质，以最大限度地覆盖人类PA蛋白质组。
