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 C18 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.

中文翻译：

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用于评估组蛋白修饰分析的中下蛋白质组学工作流程性能的要点

中下蛋白质组学已成为研究组合组蛋白翻译后修饰 (PTM) 的首选方法。在常见的自下而上的工作流程中，组蛋白被消化成相对短的肽（4-20 个氨基酸），使用反相色谱法进行分离，并使用质谱中的典型蛋白质组学方法进行分析。在中下，组蛋白被切割成较长的多肽（50-60 个氨基酸），主要对应于其 N 末端尾部，使用弱阳离子交换-亲水相互作用液相色谱（WCX-HILIC）进行解析，并使用不太传统的质谱法进行分析，即使用电子转移解离 (ETD) 进行分析物碎裂。中下法在 PTM 分析中的应用程度远不如自下而上法，部分原因是其重现性和稳健性有限。这也限制了区分好与差质量实验的严格基准的建立。在这里，我们描述了中下工作流程的关键方面，以帮助用户评估是否存在偏见和误导性结果。具体来说，我们测试了脱盐步骤中多孔石墨碳 (PGC) 的使用，证明仅使用 C18 材料脱盐会导致样品损失。我们还测试了 WCX-HILIC 缓冲液中的不同盐对组蛋白尾片段变体分析的保留、选择性和重现性的影响，特别是在缓冲液 A 中用乙二铵离子替换铵离子。这些取代对选择性和重复性有显着影响。保留。我们的结果提供了一种简化的方法来评估中下性能，以识别和量化组合组蛋白 PTM。