Department of Chemistry, University of Wisconsin-Madison.
Department of Chemistry, University of Wisconsin-Madison; School of Pharmacy, University of Wisconsin-Madison;
J Vis Exp. 2022 May 4(183). doi: 10.3791/63735.
Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological matrices is often necessary due to their low stoichiometry in comparison to non-modified analytes. Most enrichment workflows of PTMs on peptides in bottom-up proteomics workflows, where proteins are enzymatically digested before the resulting peptides are analyzed, only enrich one type of modification. It is the entire complement of PTMs, however, that leads to biological functions, and enrichment of a single type of PTM may miss such crosstalk of PTMs. PTM crosstalk has been observed between protein glycosylation and phosphorylation, the two most common PTMs in human proteins and also the two most studied PTMs using mass spectrometry workflows. Using the simultaneous enrichment strategy described herein, both PTMs are enriched from post-mortem human pancreatic tissue, a complex biological matrix. Dual-functional Ti(IV)-immobilized metal affinity chromatography is used to separate various forms of glycosylation and phosphorylation simultaneously in multiple fractions in a convenient spin tip-based method, allowing downstream analyses of potential PTM crosstalk interactions. This enrichment workflow for glyco- and phosphopeptides can be applied to various sample types to achieve deep profiling of multiple PTMs and identify potential target molecules for future studies.
质谱分析可以提供广泛的翻译后修饰（PTM）覆盖范围，尽管与非修饰的分析物相比，这些修饰物的化学计量比低，因此通常需要从复杂的生物基质中富集这些修饰物。在从头蛋白质组学工作流程中，大多数肽段的 PTM 富集工作流程中，蛋白质在分析前通过酶消化，仅富集一种类型的修饰物。然而，正是整个 PTM 复合物导致了生物学功能，而且富集单一类型的 PTM 可能会错过这种 PTM 之间的串扰。PTM 串扰已经在蛋白质糖基化和磷酸化之间观察到，这两种是人类蛋白质中最常见的 PTM，也是使用质谱工作流程研究最多的两种 PTM。本文描述的同时富集策略可从死后的人类胰腺组织（一种复杂的生物基质）中富集这两种 PTM。双功能 Ti（IV）固定化金属亲和层析可用于在方便的基于 spin tip 的方法中同时将多种形式的糖基化和磷酸化在多个馏分中分离，从而对潜在的 PTM 串扰相互作用进行下游分析。这种用于糖肽和磷酸肽的富集工作流程可应用于各种样品类型，以实现多种 PTM 的深度分析，并确定未来研究的潜在靶分子。
