Melo-Braga Marcella Nunes, Ibáñez-Vea María, Larsen Martin Røssel, Kulej Katarzyna
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.
Methods Mol Biol. 2015;1295:275-92. doi: 10.1007/978-1-4939-2550-6_21.
Post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and they are associated with a range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are generally present in substoichiometric levels and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task requiring highly specialized and sensitive enrichment methods. Currently, several methods have been implemented for PTM enrichment and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for most of the more than 300 known modifications we have none or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution is subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. In addition, the acetylated peptides present in the first TiO2 flow-through are enriched by immunoprecipitation (IP). Finally, the samples are fractionated by hydrophilic interaction liquid chromatography (HILIC) to reduce sample complexity and increase the coverage during LC-MS/MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.
翻译后修饰（PTM），如磷酸化、乙酰化和糖基化，是蛋白质功能的重要调节机制，它们与一系列生物过程相关。由于大多数翻译后修饰会改变蛋白质的分子量，质谱（MS）是研究各种翻译后修饰的理想分析工具。然而，翻译后修饰通常以亚化学计量水平存在，因此其未修饰的对应物往往会在质谱中抑制它们的信号。因此，通过质谱进行翻译后修饰分析是一项具有挑战性的任务，需要高度专业化和灵敏的富集方法。目前，已经实施了几种用于翻译后修饰富集的方法，每种方法都有其缺点和优点，因为它们对特定蛋白质修饰的选择性和特异性不同。不幸的是，对于300多种已知修饰中的大多数，我们没有或只有很差的选择性富集工具。在此，我们描述了一种全面的工作流程，用于同时研究来自同一生物样品的磷酸化、乙酰化和N-连接唾液酸化糖基化。该方案包括一个初始的二氧化钛（TiO2）步骤，用于富集磷酸肽和唾液酸化N-连接糖肽，然后进行聚糖释放和分级分离，使用固定金属亲和色谱（SIMAC）的顺序洗脱将单磷酸化肽和去糖基化肽与多磷酸化肽分离。随后，将IMAC的流出物和酸性洗脱物进行下一轮TiO2富集，以进一步将单磷酸肽与去糖基化肽分离。此外，第一次TiO2流出物中存在的乙酰化肽通过免疫沉淀（IP）进行富集。最后，通过亲水相互作用液相色谱（HILIC）对样品进行分级分离，以降低样品复杂性并增加液相色谱-串联质谱（LC-MS/MS）分析期间的覆盖范围。这允许从同一高度复杂的生物样品中分析多种类型的修饰，而不会降低每个单独的翻译后修饰研究的质量。
