Zhao Yanqing, Xu Wenhui, Jia Qiong
College of Chemistry, Jilin University, Changchun 130012, China.
Se Pu. 2022 Oct;40(10):862-871. doi: 10.3724/SP.J.1123.2022.06026.
Phosphorylation and glycosylation of proteins, two of the most widely studied post-translational modifications (PTMs), have shown increasing potential in the early non-invasive diagnosis, prognosis, and therapeutic evaluation of diseases. Besides regulating the function of cell membranes and intracellular signal transduction, protein phosphorylation participates in mitochondrial function and cellular and transcriptional metabolism. Protein glycosylation plays an important role in both intracellular and extracellular signal transduction and intracellular endocytosis. Aberrant phosphorylation and glycosylation of proteins are frequently observed in clinical proteomic studies and in the discovery of disease-related biomarkers. There are generally three methods for detecting protein phosphorylation/glycosylation: isotope radiolabeling, western blotting, and mass spectrometry. Mass spectrometry has become the most important and advantageous detection method due to its high throughput and time- and labor-efficiency. However, phosphopeptides and glycopeptides have low stoichiometry and ionization efficiency, and a large number of non-phosphopeptides and -glycopeptides interference. These issues make it difficult to directly detect phosphopeptides and glycopeptides by mass spectrometry. Therefore, the enrichment of phosphopeptides and glycopeptides before mass spectrometry detection is a key step. At present, a variety of materials have been developed for enrichment studies of phosphopeptides and glycopeptides. For example, immobilized metal affinity (IMAC) and metal oxide affinity chromatography (MOAC) methods are mostly used for the enrichment of phosphopeptides. The IMAC mainly uses positively charged metal ions and negatively charged phosphate groups to attract each other for the purpose of enriching phosphopeptides. MOAC materials rely on the chelation of metal atoms and phosphate oxygens to capture phosphopeptides. IMAC and MOAC materials rely on strong interactions between metals and phosphate groups, which often lead to difficult elution. The enrichment method for glycopeptides is mainly based on the difference in hydrophilicity between glycopeptides and non-glycopeptides, which are mainly enriched by hydrophilic interaction chromatography (HILIC). In addition, materials containing compounds such as boronic acid and lectin materials are also widely used for the separation and enrichment of glycopeptides. Smart responsive materials have also been successively reported for the enrichment of phosphopeptides and glycopeptides due to their unique responsiveness and reversibility. Smart responsive materials can respond to external stimuli; undergo structural and property changes; and convert signals such as optical, electrical, thermal, and mechanical into biochemical signals. Responsive molecules are a prerequisite for determining the response properties of smart responsive materials, and their reversible isomerization under different stimuli (such as temperature, pH, light, mechanical stress, and electromagnetic field) will lead to dynamic changes in the physical and chemical properties of materials. Compared with traditional materials, smart responsive materials can be reversibly "turned on" and "off" with better controllability. Exogenous stimuli, including temperature, light, ultrasound, electromagnetic field, and mechanical stress, can be implemented in a specific time and space. Exogenous responsive materials do not depend on changes in the reaction system itself and are non-invasive. Enzymes, pH, redox, solution polarity, and ionic strength are endogenous stimuli. Endogenous responsive materials depend on changes in the reaction system itself, and sometimes the regulation process requires the introduction of other chemicals into the reaction system. The identification, capture, and release of phosphopeptides or glycopeptides can be achieved by modulating the interactions between smart responsive materials and phosphopeptides or glycopeptides (such as hydrogen bonds, and electrostatic and hydrophobic interactions). This review classifies smart responsive materials according to the types of stimuli, which are specifically divided into exogenous and endogenous responsive materials. The enrichment of phosphopeptides and glycopeptides of exogenous/endogenous responsive materials and endogenous/exogenous co-responsive materials are summarized. In addition, we discuss the development prospects of smart responsive materials in the enrichment of phosphopeptides and glycopeptides, and also raised the challenges existing in the application of smart responsive materials in other protein post-translational modifications.
蛋白质的磷酸化和糖基化是研究最为广泛的两种翻译后修饰（PTM），在疾病的早期非侵入性诊断、预后评估及治疗评价方面展现出越来越大的潜力。除了调节细胞膜功能和细胞内信号转导外，蛋白质磷酸化还参与线粒体功能以及细胞和转录代谢。蛋白质糖基化在细胞内和细胞外信号转导以及细胞内吞作用中均发挥着重要作用。在临床蛋白质组学研究以及疾病相关生物标志物的发现中，经常会观察到蛋白质的异常磷酸化和糖基化。检测蛋白质磷酸化/糖基化通常有三种方法：同位素放射性标记、蛋白质免疫印迹法和质谱分析法。由于具有高通量以及省时省力的特点，质谱分析法已成为最重要且最具优势的检测方法。然而，磷酸化肽段和糖基化肽段的化学计量比和电离效率较低，并且存在大量非磷酸化肽段和非糖基化肽段的干扰。这些问题使得通过质谱直接检测磷酸化肽段和糖基化肽段变得困难。因此，在质谱检测之前对磷酸化肽段和糖基化肽段进行富集是关键步骤。目前，已开发出多种用于磷酸化肽段和糖基化肽段富集研究的材料。例如，固定化金属亲和（IMAC）和金属氧化物亲和色谱（MOAC）方法大多用于磷酸化肽段的富集。IMAC主要利用带正电荷的金属离子和带负电荷的磷酸基团相互吸引来富集磷酸化肽段。MOAC材料依靠金属原子与磷酸氧的螯合作用来捕获磷酸化肽段。IMAC和MOAC材料依赖于金属与磷酸基团之间的强相互作用，这常常导致洗脱困难。糖基化肽段的富集方法主要基于糖基化肽段与非糖基化肽段在亲水性上的差异，主要通过亲水作用色谱（HILIC）进行富集。此外，含有硼酸等化合物的材料以及凝集素材料也被广泛用于糖基化肽段的分离和富集。由于具有独特的响应性和可逆性，智能响应材料也相继被报道用于磷酸化肽段和糖基化肽段的富集。智能响应材料能够对外界刺激做出响应；发生结构和性质变化；并将光、电、热和机械等信号转化为生化信号。响应分子是决定智能响应材料响应特性的前提条件，其在不同刺激（如温度、pH值、光、机械应力和电磁场）下的可逆异构化会导致材料物理和化学性质的动态变化。与传统材料相比，智能响应材料可以更好地可控地可逆“开启”和“关闭”。包括温度、光、超声、电磁场和机械应力在内的外源性刺激可以在特定的时间和空间内实现。外源性响应材料不依赖于反应体系本身的变化，并且是非侵入性的。酶、pH值、氧化还原、溶液极性和离子强度是内源性刺激。内源性响应材料依赖于反应体系本身的变化，有时调节过程需要向反应体系中引入其他化学物质。通过调节智能响应材料与磷酸化肽段或糖基化肽段之间的相互作用（如氢键、静电和疏水相互作用），可以实现磷酸化肽段或糖基化肽段的识别、捕获和释放。本综述根据刺激类型对智能响应材料进行分类，具体分为外源性和内源性响应材料。总结了外源性/内源性响应材料以及内源性/外源性协同响应材料对磷酸化肽段和糖基化肽段的富集情况。此外，我们还讨论了智能响应材料在磷酸化肽段和糖基化肽段富集中的发展前景，同时也提出了智能响应材料在其他蛋白质翻译后修饰应用中存在的挑战。
