Tandem Mass Tag Labeling Facilitates Reversed-Phase Liquid Chromatography-Mass Spectrometry Analysis of Hydrophilic Phosphopeptides.
Tandem Mass Tag Labeling Facilitates Reversed-Phase Liquid Chromatography-Mass Spectrometry Analysis of Hydrophilic Phosphopeptides.
复制标题
串联质量标签标记有利于亲水性磷酸肽的反相液相色谱-质谱分析。
DOI：
10.1021/acs.analchem.9b01814
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发表时间：
2019
影响因子：
7.4
通讯作者：
Shi,Tujin
中科院分区：
文献类型：
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作者：
Tsai,Chia-Feng;Smith,JeffreyS;Krajewski,Krzysztof;Zhao,Rui;Moghieb,AhmedM;Nicora,CarrieD;Xiong,Xinyu;Moore,RonaldJ;Liu,Tao;Smith,RichardD;Jacobs,JonM;Rajagopal,Sudarshan;Shi,Tujin
蛋白质磷酸化是一种关键的翻译后修饰 (PTM)。尽管最近在基于反相液相色谱 (RPLC) 的质谱 (MS) 蛋白质组学方面取得了技术进步，但复杂生物系统中全面的磷酸化蛋白质组覆盖仍然具有挑战性，特别是对于具有丰富的丝氨酸、苏氨酸和酪氨酸区域的亲水性磷酸肽，这些区域通常协调关键的生物功能。为了解决这个问题，我们开发了一种简单、易于实施的方法，引入常用的串联质量标签（TMT）来增加肽的疏水性，从而有效增强亲水肽的 RPLC-MS 分析。与传统的 TMT 标记不同，该方法利用非伯胺缓冲液，并在基于 C18 的固相萃取之前进行 TMT 标记。通过对MCF7细胞的磷酸蛋白质组学分析，我们证明该方法可以大大增加鉴定的亲水性磷酸肽的数量并改善MS检测信号。我们应用这种方法来研究肽 QPSSSR，这是一种非常亲水的胰蛋白酶肽，位于 G 蛋白偶联受体 (GPCR) CXCR3 的 C 末端。 QPSSSR的鉴定从未有报道，我们无法通过传统方法对其进行检测。我们通过对亲水性 QPSSSR 肽库和常见磷酸肽进行比较 RPLC-MS 分析来验证我们的 TMT 标记策略。我们通过在预测会改变 QPSSSR 磷酸化的不同处理条件下量化 HEK 293 细胞中的 QPSSSR 磷酸化丰度，进一步证实了该方法的实用性。我们预计这种简单的 TMT 标记方法不仅可广泛用于解码 GPCR 磷酸蛋白质组，还可用于其他高亲水性分析物的有效 RPLC-MS 分析。
Protein phosphorylation is a critical post-translational modification (PTM). Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides with enriched regions of serines, threonines, and tyrosines that often orchestrate critical biological functions. To address this issue, we developed a simple, easily implemented method to introduce a commonly used tandem mass tag (TMT) to increase peptide hydrophobicity, effectively enhancing RPLC-MS analysis of hydrophilic peptides. Different from conventional TMT labeling, this method capitalizes on using a nonprimary amine buffer and TMT labeling occurring before C18-based solid phase extraction. Through phosphoproteomic analyses of MCF7 cells, we have demonstrated that this method can greatly increase the number of identified hydrophilic phosphopeptides and improve MS detection signals. We applied this method to study the peptide QPSSSR, a very hydrophilic tryptic peptide located on the C-terminus of the G protein-coupled receptor (GPCR) CXCR3. Identification of QPSSSR has never been reported, and we were unable to detect it by traditional methods. We validated our TMT labeling strategy by comparative RPLC-MS analyses of both a hydrophilic QPSSSR peptide library as well as common phosphopeptides. We further confirmed the utility of this method by quantifying QPSSSR phosphorylation abundances in HEK 293 cells under different treatment conditions predicted to alter QPSSSR phosphorylation. We anticipate that this simple TMT labeling method can be broadly used not only for decoding GPCR phosphoproteome but also for effective RPLC-MS analysis of other highly hydrophilic analytes.