基于还原胺化反应的氨基修饰复合纳米材料高选择性糖蛋白质固相富集质谱鉴定新方法研究

As one of the most ubiquitous and most complicated post-translation modifications of proteins, glycosylation has been estimated to be involved in more than half of cancer biomarkers. However, due to the low abundance as well as the low ionization efficiency of glycopeptides, the profiling of glycosylation has often been restrained by the signal suppression of coexisting non-glycosylated peptides in mass spectra. As a consequence, the pre-fractionation of glycoproteins/glycopeptides from complex biological samples with high specificity and high sensitivity is the necessary prerequisite for mass spectrometry analysis. Currently, continuous efforts have been devoted to develop strategies for isolation of glycopetides/glycoproteins from complex biological samples, while any of them are far from perfect. For example, due to the unsurpassable specificity of solid phase extraction by hydrazide chemistry, it had drawn extensive attention. However, it turned out to be sample-consuming and expensive, which inevitably became the bottleneck of such strategy. In this project, we proposed to develop a novel separation technique based on the conjugation of aldehydes from oxidized glycopeptides and amine-group on the magnetic nanoparticles via reductive amination reaction. Compared with the traditional solid-phase extraction methods realized by hydrzide chemistry, the desalting step is no longer needed in this novel protocol, which facilitates the realization of higher sensitivity. In addition, this strategy could render excellent enrichment performance on the premise of reducing coupling time by more than half. Thus the advantages of such strategy are obvious: on the one hand, the synthetic procedure of materials is easily accessible and cost-effective. On the other hand, the established isolation protocol is compatible for MS analysis and possesses the virtues of high sensitivity and high specificity. In this project, we especially focused on the synthesis of amine-functionalized nanoparticles，and systematically investigated the influence of modified functional groups and matrix materials on the isolation performance so as to find the optimized separation conditions. Then a brand-new protocol based on amine-functionalized magnetic nanoparticles for glycoprotein/glycopeptides enrichment combined with bio-MS technologies will be established and applied for the characterization of glycoproteome of human colorectal cancer serum.

糖基化修饰是细胞中最重要的翻译后修饰之一，超过一半的癌症生物标志物都是糖蛋白质。由于糖蛋白丰度较低且在质谱中易受非修饰蛋白信号抑制，从复杂样品中高灵敏、高选择性地富集糖蛋白已成为它们能否被质谱高灵敏分析的瓶颈。目前选择性最高的糖蛋白富集方法是基于肼腙反应的固相富集，但该方法尚存在富集材料价格贵、灵敏度低、富集时间长等问题。项目拟在前期基础上，提出基于糖蛋白中糖链上羟基氧化后产生醛基和氨基修饰材料上氨基间的还原胺化反应发展新型的糖蛋白固相富集技术。由于还原胺化反应前无需对氧化的糖蛋白除盐，该方法比肼腙法灵敏度更高，还原胺化反应时间也比肼腙反应缩短一半以上，因此新方法将具有：材料制备简单、选择性好、灵敏度高、速度快等特点。项目拟重点研究氨基修饰材料制备，探索修饰分子类型和基体材料对富集效率的影响及优化最佳富集条件，进而建立一种新型糖蛋白固相富集质谱鉴定的方法，并应用于人大肠癌血清糖蛋白组研究。

糖基化修饰是细胞中最重要的翻译后修饰之一，超过一半的癌症生物标志物都是糖蛋白质。由于糖蛋白丰度较低且在质谱中易受非修饰蛋白信号抑制，从复杂样品中高灵敏、高选择性地富集糖蛋白已成为它们能否被质谱高灵敏分析的瓶颈。目前选择性最高的糖 蛋白富集方法是基于肼腙反应的固相富集，但该方法尚存在富集材料价格贵、灵敏度低、富集时间长等问题。项目在前期基础上，提出了基于糖蛋白中糖链上羟基氧化后产生醛基和氧氨基修饰材料上氧氨基间的肟点击胺化反应，或者和苯胺修饰材料上的苯胺基团发展非还原胺化反应，发展新型的糖蛋白固相富集技术。肟点击反应，速度快，富集时间比肼腙反应缩短一半以上；非还原胺化反应不需要还原剂辅助，进一步提高里灵敏度。在研究的基础上，进一步针对糖蛋白质结合的N-糖链开展了高效衍生和高灵敏离子化研究，将糖链的离子化效率提高了30倍以上，实现了N-糖链的准确定性定量分析，定量在2个数量级范围内，呈现良好的准确性（CV<20%）在并应用于人大肠癌血清糖蛋白组研究，发现在大肠癌病人血清中13条糖链呈上调趋势，且多数具有复杂型和平分型结构。在这些高表达的糖链中，有4条是核心岩藻糖化的，2条是唾液酸化的，该结果与前期文献报道的在肠癌发展过程中，N-糖链分支变多且核心岩藻糖化、唾液酸化水平增加起重要作用的结论相一致。