Doan Ngoc-Duc, DiChiara Andrew S, Del Rosario Amanda M, Schiavoni Richard P, Shoulders Matthew D
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA.
Koch Institute, Massachusetts Institute of Technology, Cambridge, MA, USA.
Methods Mol Biol. 2019;1944:95-114. doi: 10.1007/978-1-4939-9095-5_7.
We present the development, optimization, and application of constructs, cell lines, covalent cross-linking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome cross-linking and perform immunoprecipitations are then presented. We conclude with a discussion of methods to elute collagen interactomes and prepare samples for mass spectrometry-mediated identification of interactors. Throughout, caveats and potential problems researchers may encounter when working with collagen are discussed. We note that the protocols presented herein may be readily adapted to define interactomes of other collagen types, as well as to determine comparative interactomes of normal and disease-causing collagen variants using quantitative isotopic labeling (SILAC)- or isobaric mass tags (iTRAQ or TMT)-based mass spectrometry analysis.
我们展示了构建体、细胞系、共价交联方法和免疫沉淀策略的开发、优化及应用，这些能够通过基于质谱的蛋白质组学实现对胶原蛋白相互作用组的稳健且准确的测定。以I型胶原蛋白为例，描述了处理大型、重复且富含GC的胶原蛋白基因的方案，接着介绍了稳定且可诱导表达抗体表位标签化I型胶原蛋白的细胞工程策略。随后给出了优化胶原蛋白相互作用组交联及进行免疫沉淀的详细步骤。我们最后讨论了洗脱胶原蛋白相互作用组并制备样品以用于质谱介导的相互作用蛋白鉴定的方法。贯穿全文，还讨论了研究人员在处理胶原蛋白时可能遇到的注意事项和潜在问题。我们指出，本文介绍的方案可轻松适用于定义其他类型胶原蛋白的相互作用组，以及使用基于定量同位素标记（SILAC）或等压质量标签（iTRAQ或TMT）的质谱分析来确定正常和致病胶原蛋白变体的比较相互作用组。
