development and validation of a method for profiling post-translational modification activities using protein microarrays开发和验证方法使用蛋白质微阵列分析翻译修饰活动.pdfVIP

Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays ´ 1. 2. 3 1 1 Sonia V. del Rincon , Jeff Rogers , Martin Widschwendter , Dahui Sun , Hans B. Sieburg , Charles Spruck1* 1 Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America, 2 Life Technologies Corporation, Carlsbad, California, United States of America, 3 Department of Gynaecological Oncology, University College London, London, United Kingdom Abstract Background: Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens. Methodology/Principal Findings: In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile t