摘要
蛋白质的N-糖基化修饰在多种生物过程中发挥着至关重要的作用,近年来的许多研究证实异常的蛋白质糖基化与多种疾病的发生发展密切相关,表明糖基化蛋白质具有较大的潜力成为新的生物标志物或者药物靶标。在样本的处理过程中,对N-糖基化肽段进行富集分离后再进行质谱分析已经成为糖蛋白质组学分析前的必要步骤。但是,由于复杂生物样本中N-糖基化肽段的丰度低和离子化效率差等问题,通过质谱鉴定N-糖肽仍然是一项艰巨的任务。研究通过将纳米金线(Au)、4-巯基苯硼酸(4-MPB)与超薄二维二硫化钼(2D-MoS2)进行反应,成功制备了一种用于富集蛋白质N-糖基化肽段的新型功能纳米复合材料(MoS2/Au/4-MPB)。二硫化钼纳米材料的层状结构可以为反应提供大量的可修饰位点,便于修饰纳米金线;功能基团4-巯基苯基硼酸对N-糖肽具有高度的选择性,可以对生物样品中N-糖基化肽段进行特异性富集。使用标准蛋白人免疫球蛋白G(IgG)和牛血清白蛋白(BSA)胰蛋白酶酶切产物对新型功能纳米材料的N-糖基化肽段的富集性能进行评估,其灵敏度达到5 fmol,选择性达到1∶1000。将其用于生物样品中N-糖基化肽段的富集,从50μg尿液外泌体蛋白胰蛋白酶酶切产物中共富集鉴定出768个N-糖肽,归属于377个蛋白质。这些结果表明该新型功能纳米复合材料对复杂生物样品中N-糖肽的选择富集有着巨大的应用潜力,为糖蛋白质组的研究提供了一种新方法。
Protein N-glycosylation plays a crucial role in the folding,transportation,and localization of proteins,and participates in many important biological processes such as receptor activation and signal transduction.An increasing number of studies have shown that abnormal protein glycosylation is closely related to various diseases.Therefore,N-glycosylated proteins are potential candidates for become new biomarkers or drug targets.The current research strategy for N-glycosylated proteins is to first digest the protease into peptides and then identify them by mass spectrometry.In sample preparation processing,enrichment and separation of N-glycopeptides have become the vital step for glycoproteomic analysis.However,because of their low abundance and poor ionization,mass spectrometric identification of N-glycopeptides in complex samples remains a challenging task.Therefore,the development of a selective enrichment strategy for N-glycopeptides from complex biological samples is necessary.In this work,new functional nanomaterials were prepared through a simple,convenient,and efficient two-step“Au-S”reaction.In short,the“Au-S”bond was first used to load nano-gold wires on the surface of molybdenum disulfide(MoS2),and then to connect these wires to 4-mercaptophenylboronic acid(4-MPB).The resulting nanomaterial named MoS2/Au/4-MPB was successfully prepared by serial functionalization of ultra-thin two-dimensional MoS2,nano-gold wires and 4-MPB for the efficient enrichment of N-glycopeptides.The layered structure of molybdenum disulfide nanomaterials can provide numerous modifiable sites for the reaction,allowing for convenient modification of the nano-gold wires.The functional group 4-MPB has high affinity for N-glycopeptides,and it can selectively enrich them in biological samples.In order to evaluate the N-glycopeptide enrichment performance of MoS2/Au/4-MPB,standard proteins human immunoglobulin G(IgG)and bovine serum albumin(BSA)trypsin digests were used.Thus,facilitated interactions with N-glycopeptides for boric acid-based retention could be expected.Low femtomolar detection sensitivity,1∶1000 enrichment selectivity,and 100μg/mg loading capacity were achieved for N-glycopeptide enrichment.In the application of this material to biological samples,exosomes were chosen as the research objects.On the one hand,exosomes serve as a new class of carriers for intercellular communication and drug delivery.Almost all types of cells can secrete exosomes.On the other hand,urine is another potential source for liquid biopsy samples in addition to blood.Obtaining urine samples is a less invasive process,and urine samples are easier to handle and save as compared to blood samples.Exosomes can also be detected in urine.Cell-specific components such as proteins,lipids,and nucleic acids can provide diverse information,which is of great significance in disease diagnosis and treatment.However,there are only a few studies on urinary exosomes N-glycoprotein group.In this research,N-glycoproteins were successfully identified in urine exosomes.In three replicate experiments,536,515 and 487 N-glycosylated peptides were identified from 279,270 and 279 N-glycoproteins,respectively.After de-duplication of the three sets of data,a total of 768 N-glycopeptides corresponding to 377 N-glycoproteins were identified.This result indicates that the novel nanocomposite has good enrichment selectivity and sensitivity for N-glycopeptides enrichment in complex biological samples,and this method provides a new method for glycoproteomics research.
作者
张汉卿
秦伟捷
张养军
ZHANG Hanqing;QIN Weijie;ZHANG Yangjun(Graduate School, Anhui Medical University, Hefei 230032, China;Beijing Institute of Lifeomics, Beijing Proteome Research Center, State Key Laboratory of Proteomics, Beijing 102206, China)
出处
《色谱》
CAS
CSCD
北大核心
2020年第8期891-899,共9页
Chinese Journal of Chromatography
基金
国家重点研发计划(2017YFA0505002,2018YFC0910302,2016YFA0501403,2018YFF0212505)
国家自然科学基金(21675172)
蛋白质组学国家重点实验室(SKLP-K201706)
北京市泌尿细胞分子诊断重点实验室(2019-KF21)。
关键词
富集
二硫化钼
4-巯基苯硼酸
外泌体
N-糖基化肽段
enrichment
molybdenum disulfide
4-mercaptophenylboronic acid
exosomes
N-glycopeptides
