Sulfotyrosine and phosphotyrosine are two post-translational modifications present in higher eukaryotes. A simple and direct mass spectrometry method to distinguish between these modifications is crucial to advance our understanding of the sulfoproteome. While sulfation and phosphorylation are nominally isobaric, the accurate mass of the sulfuryl moiety is 9.6 mDa less than the phosphoryl moiety. Based on this difference, we have used an Orbitrap Fusion Lumos mass spectrometer to characterize, resolve, and distinguish between sulfotyrosine and phosphotyrosine modifications using a set of model peptides. Multiple fragmentation techniques, namely HCD, CID, ETD, ETciD, and EThcD, have been used to compare the different fragmentation behaviors between peptides modified with these species. Sulfotyrosine undergoes neutral loss using HCD and CID, but the sulfuryl moiety is largely stable under ETD. In contrast, phosphotyrosine is stable during fragmentation using all these methods. This differential stability provides a mechanism to distinguish sulfopeptides from phosphopeptides. Based on the rigorous characterization presented herein, this work serves as a model for accurate identification of phosphotyrosine and, more challenging, sulfotyrosine, in complex proteomic samples.

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磺基酪氨酸和磷酸酪氨酸是高级真核生物中存在的两个翻译后修饰。区分这些修饰的简单直接的质谱方法对于增进我们对磺基蛋白质组学的理解至关重要。尽管硫酸化和磷酸化名义上是等压的，但磺酰基部分的准确质量比磷酰基部分小9.6 mDa。基于此差异，我们使用Orbitrap Fusion Lumos质谱仪来表征，分辨和区分使用一组模型肽的磺基酪氨酸和磷酸酪氨酸修饰。已使用多种片段化技术（即HCD，CID，ETD，ETciD和EThcD）来比较用这些物种修饰的肽之间的不同片段化行为。硫代酪氨酸通过HCD和CID受到中性损失，但是亚磺酰基部分在ETD下基本稳定。相反，使用所有这些方法在断裂过程中磷酸酪氨酸是稳定的。这种差异稳定性提供了将磺肽与磷酸肽区分开的机制。基于本文提出的严格表征，这项工作可作为准确鉴定复杂蛋白质组学样品中磷酸酪氨酸以及更具挑战性的磺基酪氨酸的模型。

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