DeHart Caroline J, Fellers Ryan T, Fornelli Luca, Kelleher Neil L, Thomas Paul M
Departments of Chemistry, Molecular Biosciences, the Proteomics Center of Excellence and the Robert H. Lurie Comprehensive Cancer Center at the Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA.
Methods Mol Biol. 2017;1558:381-394. doi: 10.1007/978-1-4939-6783-4_18.
Traditional bottom-up mass spectrometry-based proteomics relies on the use of an enzyme, often trypsin, to generate small peptides (typically < 25 amino acids long). In top-down proteomics, proteins remain intact and are directly measured within the mass spectrometer. This technique, while inherently simpler than bottom-up proteomics, generates data which must be processed and analyzed using software tools "purpose-built" for the job. In this chapter, we will show the analysis of intact protein spectra through deconvolution, deisotoping, and searching with ProSight Lite, a free, vendor-agnostic tool for the analysis of top-down mass spectrometry data. We will illustrate with two examples of intact protein fragmentation spectra and discuss the iterative use of the software to characterize proteoforms and discover the sites of post-translational modifications.
传统的基于自下而上质谱的蛋白质组学依赖于使用一种酶（通常是胰蛋白酶）来生成小肽（通常长度小于25个氨基酸）。在自上而下的蛋白质组学中，蛋白质保持完整，并在质谱仪中直接进行测量。这项技术虽然本质上比自下而上的蛋白质组学更简单，但生成的数据必须使用专门为此任务“量身定制”的软件工具进行处理和分析。在本章中，我们将展示通过去卷积、去同位素以及使用ProSight Lite进行搜索来分析完整蛋白质谱，ProSight Lite是一款免费的、与供应商无关的用于分析自上而下质谱数据的工具。我们将通过两个完整蛋白质片段化谱的例子进行说明，并讨论该软件的迭代使用，以表征蛋白质变体并发现翻译后修饰位点。
