Sulfation is considered the most prevalent post-translational modification (PTM) on tyrosine; however, its importance is frequently undervalued due to difficulties in direct and unambiguous determination from phosphorylation. Here we present a sequence-independent strategy to directly map and quantify the tyrosine sulfation states in universal native peptides using an engineered protein nanopore. Molecular dynamics simulations and nanopore mutations reveal specific interactions between tyrosine sulfation and the engineered nanopore, dominating identification across diverse peptide sequences. We show a nanopore framework to discover tyrosine sulfation in unknown peptide fragments digested from a native protein and determine the sequence of the sulfated fragment based on current blockade enhancement induced by sulfation. Moreover, our method allows direct observation of peptide sulfation in ultra-low abundance, down to 1%, and distinguishes it from isobaric phosphorylation. This sequence-independent strategy suggests the potential of nanopore to explore specific PTMs in real-life samples and at the omics level.

硫酸化被认为是最常见的酪氨酸翻译后修饰 (PTM)；然而，由于直接和明确地测定磷酸化存在困难，其重要性经常被低估。在这里，我们提出了一种独立于序列的策略，使用工程蛋白纳米孔直接绘制和量化通用天然肽中的酪氨酸硫酸化状态。分子动力学模拟和纳米孔突变揭示了酪氨酸硫酸化和工程纳米孔之间的特定相互作用，主导了不同肽序列的识别。我们展示了一个纳米孔框架，用于发现从天然蛋白质消化的未知肽片段中的酪氨酸硫酸化，并根据硫酸化诱导的电流阻断增强确定硫酸化片段的序列。此外，我们的方法可以直接观察超低丰度（低至 1%）的肽硫酸化，并将其与同量异位磷酸化区分开来。这种与序列无关的策略表明纳米孔在现实样品和组学水平上探索特定 PTM 的潜力。