基于非特异性酶切同位素标记糖链相对定量新方法的研究及在癌症生物标志物筛选中的应用

The lack of highly sensitive, and simple method for quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. Because of the complexity of the structure of the glycan’s, the non-uniformity of their micro-structures, and the difficulty of synthesizing the standard glycan’s hinder the development of method determining glycan’s quantitatively. Chemical labeling combined with mass spectrometry (MS) detecting glycan’s is a potential method of analyzing glycan’s quantitatively. In this study, we will use electrospray ionization mass spectrometry (ESI-MS), based on non-specific Pronase E, which will hydrolyze a glycoprotein to an glycosylamino acid carrying one amino acid, to analyze N-/O-glycan. A novel, derivative reagent, which carries the positive charge, with both light and heavy isotopes, will be designed and synthesized. With the usage of this derivative reagent, it will be easier to infer the micro-structures of the fragments from glycan’s in mass spectrometry, comparing to the traditional methods. To establish a novel relative quantitative method for analyzing the N-/O-glycan’s, which can not only complete the isotopic labeling of the glycan, but also keep the original structure of the glycan. We will investigate the effect of glycan labeling using sialylglycopeptide (SGP); and also will summarize the fragmentation pattern of the oligosaccharides in the mass spectrometry. In addition, we will validate linearity, reproducibility and accuracy, using model glycopeptides. Through this study, we will provides some novel ideas with some evidence about developing quantitative analysis method of glycomics. As a pre-clinic trial, we will screen and identify the potential glycan biomarkers of colorectal cancer in early-stage. The gycosylation pattern of these glycan biomarkers will be summarized in the end. In summary, a novel screening method for identifying glycan biomarkers of cancers in early-stage and differences of glycomics will be provided by our group.

高灵敏、简便的糖链定量分析方法是蛋白质糖基化研究和评价抗体药物稳定性以及筛选疾病糖链生物标志物的瓶颈。由于糖链结构复杂和微观不均一性,且标准品难以合成，阻碍了糖链定量分析方法研究的发展。化学标记结合质谱检测是进行糖链定量分析的有效手段。鉴于此，本研究中将采用电喷雾电离质谱，基于非特异性链酶蛋白酶E，将糖蛋白酶解为带有一个氨基酸的糖氨酸,以N-/O-糖链为分析对象，设计合成带有正电荷、易于结构推断、具有轻重稳定同位素结构的新型高灵敏度质谱试剂。建立既能完成糖链的同位素标记，又能保持糖链原有结构的N-/O-糖链同时相对定量分析新方法。利用模型糖肽考察影响糖链标记效果，及在质谱中的裂解规律, 验证动态范围和准确性。为开发糖组学定量新分析方法提供思路和理论依据。并应用于早期大肠癌潜在糖链生物标志物的筛选及鉴定，考察糖基化修饰规律。为筛选癌症早期有效的糖链生物标志物及差异性糖组学提供有效的新方法。

糖链的相对定量研究对糖链生物标志物的筛选及临床有着重要的意义。但因糖链的结构复杂多样和微观不均一性，加之标准品难以合成，阻碍了糖链定量分析方法研究的发展。鉴于此，为了高灵敏度检测及定量糖链，我们开发了具有带正电荷结构、能提高质谱检测灵敏度、易于结构推断的8种稳定轻、重同位素标记糖链相对定量质谱衍生化试剂。基于同位素标记建立了糖链高灵敏度检测及相对定量分析方法。. 首先，利用同位素标记d0(d5)-DPBOC衍生化基于链霉蛋白酶E酶切建立了糖链相对定量新方法。唾液酸糖肽 SGP为糖链模型，d0(d5)同位素标记的糖链以理论摩尔比值为横坐标，实测丰度比值为纵坐标(d0/d5=20-0.029或d5/d0=20-0.029)时，d0/d5和d5/d0的标准曲线的R2=0.9994, 0.9996得到良好的线性关系，定量限为15 fmol。并成功定量了不同比例的核糖核酸酶B中的5种糖链。其次，建立了基于带正电荷4-CEBTPP衍生化糖链LC-ESI-MS/MS高灵敏度检测新方法。利用D-葡糖胺作为模型糖时可得到m/z=120的4-CEBTPP 试剂本身特征碎片离子，检测限为0.3 amol。分析唾液酸糖肽SGP时检测限为30 fmol。另外，衍生化的糖链在CE=40eV时可以得到m/z 627的4-CEBTPP-GlcNAc特征碎片离子。母离子扫描应用于核糖核酸酶B和胎球蛋白，以2价离子各检测出5种和7种糖链。最后，基于糖链转移突变体Endo-M-N175Q酶的糖链转移反应开发了既能同位素标记，又能保持糖链原有结构的糖链相对定量分析方法。利用唾液酸糖肽SGP时，得到d0/d8和d8/d0的标准曲线的R2=0.9999, 0.9978线性关系良好。另外，在人体血液中相对定量和检测到15种糖链。. 新合成的轻、重同位素标记8种糖链质谱试剂，可靶向识别和同位素标记糖链，提高糖链的疏水性，可在廉价的反相色谱柱中提高糖链检测灵敏度，并能利用同位素比值进行糖链的相对定量。本研究开发的基于高灵敏度同位素标记糖链相对定量方法可应用于人体血液等生物样品，为各种癌症等疾病糖链生物标志物的筛选提供了一种新的分析方法。