Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA; Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA; Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Methods. 2020 Dec 1;184:86-92. doi: 10.1016/j.ymeth.2020.01.013. Epub 2020 Feb 15.
Middle-down proteomics has emerged as the method of choice to study combinatorial histone post translational modifications (PTMs). In the common bottom-up workflow, histones are digested into relatively short peptides (4-20 aa), separated using reversed-phase chromatography and analyzed using typical proteomics methods in mass spectrometry. In middle-down, histones are cleaved into longer polypeptides (50-60 aa) mostly corresponding to their N-terminal tails, resolved using weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) and analyzed with less conventional mass spectrometry, i.e. using Electron Transfer Dissociation (ETD) for analyte fragmentation. Middle-down is not nearly as utilized as bottom-up for PTM analysis, partially due to its limited reproducibility and robustness. This has also limited the establishment of rigorous benchmarks to discriminate good vs poor quality experiments. Here, we describe critical aspects of the middle-down workflow to assist the user in evaluating the presence of biased and misleading results. Specifically, we tested the use of porous graphitic carbon (PGC) during the desalting step, demonstrating that desalting using only C material leads to sample loss. We also tested different salts in the WCX-HILIC buffers for their effect on retention, selectivity, and reproducibility of analysis of variants of histone tail fragments, in particular replacing ammonium ion with ethylenediammonium ion in buffer A. These substitutions had marked effects on selectivity and retention. Our results provide a streamlined way to evaluate middle-down performance to identify and quantify combinatorial histone PTMs.
中下位蛋白质组学已成为研究组合组蛋白翻译后修饰（PTMs）的首选方法。在常见的自下而上工作流程中，组蛋白被消化成相对较短的肽（4-20 个氨基酸），使用反相色谱法分离，并使用典型的质谱蛋白质组学方法进行分析。在中下位，组蛋白被切割成长度更长的多肽（50-60 个氨基酸），主要对应其 N 端尾部，使用弱阳离子交换-亲水性相互作用液相色谱（WCX-HILIC）进行分离，并使用不太常规的质谱法进行分析，即使用电子转移解离（ETD）进行分析物碎片化。中下位在 PTM 分析中的应用远不及自下而上，部分原因是其重现性和稳健性有限。这也限制了建立严格的基准来区分高质量和低质量实验。在这里，我们描述了中下位工作流程的关键方面，以帮助用户评估是否存在有偏差和误导性的结果。具体来说，我们在脱盐步骤中测试了使用多孔石墨碳（PGC），证明仅使用 C 材料进行脱盐会导致样品损失。我们还测试了 WCX-HILIC 缓冲液中不同盐对保留、选择性和分析组蛋白尾部片段变体重现性的影响，特别是在缓冲液 A 中用乙二胺代替铵离子。这些取代对选择性和保留有显著影响。我们的结果提供了一种简化的方法来评估中下位的性能，以识别和定量组合组蛋白 PTMs。
