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The Key Role of Metal Adducts in the Differentiation of Phosphopeptide from Sulfopeptide Sequences by High-Resolution Mass Spectrometry
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-06-17 , DOI: 10.1021/acs.analchem.1c05621
Susy Piovesana 1 , Anna Laura Capriotti 1 , Chiara Cavaliere 1 , Andrea Cerrato 1 , Carmela Maria Montone 1 , Riccardo Zenezini Chiozzi 2, 3 , Aldo Laganà 1
Affiliation  

Site localization of protein sulfation by high-throughput proteomics remains challenging despite the technological improvements. In this study, sequence analysis and site localization of sulfation in tryptic peptides were determined under a conventional nano-liquid chromatography-mass spectrometry configuration. Tryptic sulfopeptide standards were used to study different fragmentation strategies, including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD), and electron-transfer/collision-induced dissociation (ETciD), in the positive ionization mode. Sulfopeptides displayed only neutral loss of SO3 under CID, while the sequence could be determined for all other tested fragmentation techniques. Results were compared to the same sequences with phosphotyrosine, indicating important differences, as the sequence and modification localization could be studied by all fragmentation strategies. However, the use of metal adducts, especially potassium, provided valuable information for sulfopeptide localization in ETD and ETD-hybrid strategies by stabilizing the modification and increasing the charge state of sulfopeptides. In these conditions, both the sequence and localization could be obtained. In-source neutral loss of SO3 under EThcD provided diagnostic peaks suitable to distinguish the sulfopeptides from the nearly isobaric phosphopeptides. Further confirmation on the modification type was found in the negative ionization mode, where phosphopeptides always had the typical phosphate product ion corresponding to PO3.

中文翻译:


金属加合物在高分辨率质谱区分磷酸肽和硫肽序列中的关键作用



尽管技术有所进步,但通过高通量蛋白质组学对蛋白质硫酸化进行位点定位仍然具有挑战性。在本研究中,在传统的纳米液相色谱-质谱配置下确定了胰蛋白酶肽中硫酸化的序列分析和位点定位。胰蛋白酶磺肽标准品用于研究不同的裂解策略,包括碰撞诱导解离 (CID)、高能碰撞解离 (HCD)、电子转移解离 (ETD)、电子转移/高能碰撞解离 (EThcD)、以及正电离模式下的电子转移/碰撞诱导解离 (ETciD)。硫肽在 CID 下仅表现出 SO 3的中性损失,而可以针对所有其他测试的断裂技术来确定序列。将结果与具有磷酸酪氨酸的相同序列进行比较,表明重要的差异,因为可以通过所有片段化策略来研究序列和修饰定位。然而,金属加合物,尤其是钾的使用,通过稳定修饰和增加磺肽的电荷状态,为 ETD 和 ETD 混合策略中的磺肽定位提供了有价值的信息。在这些条件下,可以获得序列和定位。 EThcD 下 SO 3的源内中性损失提供了适合区分磺肽和近同量异序磷酸肽的诊断峰。对修饰类型的进一步确认是在负电离模式中发现的,其中磷酸肽总是具有对应于 PO 3 的典型磷酸根产物离子。
更新日期:2022-06-17
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