A Hierarchical Coding Strategy for Live Cell Imaging of Protein‐Specific Glycoform

Live cell imaging of protein‐specific glycoforms holds great promise for revolutionizing the study of glycochemistry. The imaging protocols developed thus far build upon the paired interplay of probe units, thus limiting the number of monosaccharide identification channels. A hierarchical coding (HieCo) imaging strategy, with DNA coding and decoding of protein and monosaccharides executed in fidelity to the hierarchical order of target glycoprotein, is reported herein and features expandable monosaccharide identification channels. A proof‐of‐concept protocol has been developed for MUC1‐specific imaging of terminal sialic acid (Sia) and fucose (Fuc) on MCF‐7, T47D, MDA‐MB‐231, and PANC‐1 cells, revealing distinct monosaccharide patterns for four types of cells. The protocol also permits dynamic monitoring of changes in MUC1‐specific monosaccharide patterns associated with both the alteration of cellular physiological states and the occurrence of a biologically important process.     

质谱法定量测定高密度脂蛋白结合蛋白的糖基化水平

采用质谱法对4种高密度脂蛋白(HDL)的结合蛋白重组人载脂蛋白血清淀粉样蛋白A(SAA)、 α1-抗胰蛋白酶(A1AT)、 α2-人体血清糖蛋白(A2HSG)和A载脂蛋白C3(Apo C3)从蛋白质含量(蛋白的绝对定量)、 位点特异性糖基化(糖肽的相对定量)及聚糖位点占有率等方面进行了研究. 利用四极杆-飞行时间质谱仪(Q-TOF)测量糖蛋白标样酶解产物的二级质谱碎片离子, 用Byonic软件发现了新的糖基化位点信息, 即增加了原位点处聚糖糖型的种类. 对于A2HSG, 新增了N-糖基化156位点上的4种糖型, N-糖基化176位点上的6种糖型, O-糖基化319位点的4种O-聚糖和O-糖基化346位点上的1种糖型. 对于Apo C3, 只有O-糖基化94一个位点, 在此位点上新增了9种糖型. 同时, 调整了用于定量蛋白的多肽, 使得定量更加准确. 采用三重四极杆串级质谱仪(UPLC-ESI-QQQ)研究了4种结合蛋白中多肽和糖肽的多反应监测(MRM)行为, 并重新计算了每种聚糖的位点占有率, 优化了现有的定量方法.     

蛋白质的化学糖基化研究进展

细胞内超过50%的蛋白质为糖蛋白,糖基在很大程度上影响着蛋白质的折叠、稳定性、信号传导、生物活性、免疫原性及药代动力学等.化学糖基化是获得糖基结构和糖基化位置确定的糖蛋白的有效方法.本文以糖蛋白合成技术的发展和应用为导向,围绕糖-肽键的形成,概述了蛋白的化学糖基化研究进展.     

Localized Chemical Remodeling for Live Cell Imaging of Protein-Specific Glycoform

Live cell imaging of protein-specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole-cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein-specific glycoform information is reported. The proof-of-concept protocol developed for MUC1-specific terminal galactose/N-acetylgalactosamine (Gal/GalNAc) combines affinity binding, off-on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability.     

Glycosylation analysis of interleukin-23 receptor: elucidation of glycosylation sites and characterization of attached glycan structures

Interleukin-23 (IL-23) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation. It signals through a heterodimeric receptor consisting of IL-23r, which is heavily glycosylated. The structural characterization of IL-23r has not been reported. In this work, glycosylation profiles of soluble recombinant human IL-23r (rhIL-23r) were established using mass spectrometry (MS), which included defining glycosylation sites, degree of glycosylation occupancy of each site and structure of attached oligosaccharides. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc(+)) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap) for glycosylation site profiling. The attached glycan structures were elucidated by collision-induced dissociation (CID) fragmentation of target glycopeptides in combination with accurate mass measurement. Eight glycosylation sites were identified on IL-23r (Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6). Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion. Multiple glycoforms were observed in IL-23r. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid. High mannose and hybrid type glycans were only observed on Asn157. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r.     

基于纳升电喷雾-轨道阱超高分辨质谱对完整单克隆抗体药物进行分子量测定及糖型鉴定的条件探究

整体分子量测定和糖型鉴定是抗体药物研发及生产中不可或缺的表征内容,而在完整蛋白层面进行测定可获得最为直接的测定结果。实现此类测定有赖于可提供较高分辨能力及质量精度的仪器;测定中涉及的相对复杂的参数设置可能受多种条件的干扰而产生误导性结果。该研究考察了利用纳升电喷雾离子源-轨道阱质谱仪以非变性质谱方式测定完整单克隆抗体药时,仪器分辨率、源内裂解、碰撞诱导碎裂、溶液组成等条件对单克隆抗体药物分子量及糖型测定结果的影响,以期为此类质谱分析提供有益的借鉴。