Tang Ruizhi, Yu Yang, Dong Jing, Yao Yating, Ma Shujuan, Ou Junjie, Ye Mingliang
CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China.
CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Anal Chim Acta. 2021 Feb 1;1144:111-120. doi: 10.1016/j.aca.2020.12.015. Epub 2020 Dec 11.
In bottom-up strategy, specific enrichment of glycopeptides and phosphopeptides from complicated biological samples is a prerequisite for efficient identifying glycosylation and phosphorylation by mass spectrometry. Although there were a plethora of materials used as either hydrophilic interaction liquid chromatography (HILIC) or immobilized metal affinity chromatography (IMAC) adsorbents, even several bifunctional materials for simultaneous enrichment of glycopeptides and phosphopeptides, most of them are not easily commercialized as many other well-performing adsorbents due to the complicated preparation process. In our case, a one-step modification strategy was developed to prepare bifunctional adsorbents for HILIC and IMAC, employing O-phospho-l-serine as the modifier and poly(GMA-co-EDMA) microspheres, a kind of macroporous adsorption resin (MAR) with epoxy groups, as the matrix. The MARs were directly modified with O-phospho-l-serine under facile condition for HILIC strategy and further chelated with Ti for IMAC strategy. A total of 522 unique N-glycopeptides and 442 unique N-glycosylation sites mapped to 275 N-glycoproteins was identified from HeLa cell proteins, showing excellent enrichment efficiency in HILIC. Additionally, 3141 unique phosphopeptides were unambiguously identified from 200 μg of digest of HeLa cell proteins, demonstrating great enrichment efficiency in IMAC. Moreover, these materials have been successfully applied in the analysis of multiple biological samples including human serum and milk, demonstrating their feasibility for real sample applications and potential business value.
在自下而上的策略中，从复杂生物样品中特异性富集糖肽和磷酸肽是通过质谱高效鉴定糖基化和磷酸化的前提条件。尽管有大量材料用作亲水相互作用液相色谱（HILIC）或固定金属亲和色谱（IMAC）吸附剂，甚至有几种用于同时富集糖肽和磷酸肽的双功能材料，但由于制备过程复杂，它们中的大多数并不像许多其他性能良好的吸附剂那样容易商业化。在我们的研究中，开发了一种一步修饰策略来制备用于HILIC和IMAC的双功能吸附剂，采用O-磷酸-L-丝氨酸作为修饰剂，聚（甲基丙烯酸缩水甘油酯-共-乙二甲基丙烯酸酯）微球（一种带有环氧基团的大孔吸附树脂（MAR））作为基质。在简便的条件下，MARs直接用O-磷酸-L-丝氨酸进行修饰以用于HILIC策略，并进一步与钛螯合以用于IMAC策略。从HeLa细胞蛋白质中鉴定出总共522个独特的N-糖肽和442个映射到275个N-糖蛋白的独特N-糖基化位点，在HILIC中显示出优异的富集效率。此外，从200μg的HeLa细胞蛋白质消化物中明确鉴定出3141个独特的磷酸肽，在IMAC中显示出很高的富集效率。此外，这些材料已成功应用于包括人血清和牛奶在内的多种生物样品的分析，证明了它们在实际样品应用中的可行性和潜在商业价值。
