NOVEL TAGS FOR THE STABLE ISOTOPIC LABELING OF CARBOHYDRATES

用于碳水化合物稳定同位素标记的新​​型标签

• 批准号: 7722958
• 负责人: JOSEPH ZAIA
• 金额: $ 1.94万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722958
• 关键词: Acids; Amination; Binding; Biological; Carbohydrates; Cartilage; Chondroitin ABC Lyase; Chondroitin Sulfate Proteoglycan; Class; Computer Retrieval of Information on Scientific Projects Database; Dalteparin; Enoxaparin; Enzymes; Family suidae; Funding; GAG Gene; Glycoproteins; Glycosaminoglycans; Gold; Grant; Heparin; Heparin Lyase; Institution; Ions; Label; Link; Low-Molecular-Weight Heparin; Lyase; Mass Spectrum Analysis; Methods; Molecular Sieve Chromatography; Oligosaccharides; Pattern; Peptide N-glycohydrolase F; Peptides; Performance; Pharmacologic Substance; Polysaccharides; Preparation; Proteoglycan; Pulsar; Purpose; Relative (related person); Research; Research Personnel; Resistance; Resources; Sampling; Series; Solvents; Source; Stable Isotope Labeling; Structure; System; Thrombin; United States National Institutes of Health; aggrecan; biglycan; decorin; instrument; interest; mass spectrometer; novel; progesterone 11-hemisuccinate-(2-iodohistamine); research study; stable isotope; tandem mass spectrometry

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The increasing interest in glycomics has led to the widespread use of mass spectrometry to determine the carbohydrate components expressed in biological systems. However, the quantification of carbohydrates within different samples remains a daunting task, due to instrument and sample variability. Herein, we show cusyom-synthesized multiplexed stable isotope labeled tags that have broad applicability for the simultaneous quantitation of four samples during the same mass spectrometry experiment. Results exploring the utility of multiplexed stable isotopic labeling from a variety of sources have been demonstrated. A stable isotope-labeled tag in four forms (+0,+4,+8,+12) was synthesized for the purpose of labeling the reducing end of glycans. Glycosaminoglycans (GAGs) from the chondroitin sulfate proteoglycan (CS-PG) class and pharmaceutical low-molecular weight (LMWT) heparins were partially depolymerized using enzymes to form oligosaccharide distributions. These oligosaccharides were labeled with the tetraplex stable isotope-containing tags by reductive amination. The resulting tagged GAGs were combined, separated by high performance size exclusion chromatography (Superdex peptide 3.2/30, Beckman Gold 125 solvent module), and analyzed in the negative electrospray mode using an Applied Biosystems QSTAR Pulsar-I (Q-ToF) mass spectrometer. In addition, the PNGase F released a-1-acid glycans from four species were also multiplex-labeled and analyzed. A series of glycomics tags that incorporate stable isotope modules into the oligosaccharide structure were used to label the glycans released from CS-PGs, LMWT heparins, and N-linked glycans from a-1 acid glycoproteins. The abundances of ions in the MS mode serve to quantify ions produced from a given sample in each of the four samples. These results further demonstrate the principle of quantitation by multiplex analysis of carbohydrate using stable isotope tags. Chondroitinase ABC was used to depolymerize the released GAG chains from aggrecan, biglycan, cartilage extract, and decorin. The CS-proteoglycans were normalized for concentration allowing for a mass spectrometric analysis of relative CS quantities. Additional information on the isomeric content of the CS chains was obtained via tandem mass spectral analysis utilizing the distinctive fragmentation patterns of each glycoform (CSA, CSB, and CSC). Additional experiments were performed to demonstrate greater utility of the tags in examination of GAGs. Low molecular weight heparins (Lovenox and Fragmin) were depolymerized to the greatest extent possible with heparin lyases I, II, and III. A significant fraction of heparin (and LMWT heparin) resistant to the lyase treatment should be composed of a tetrasaccharide containing a trisulfated GlcN residue that derives from the pentasaccharide sequence required for anti-thrombin III binding. The quantities of lyase resistant structures for each LMWT heparin preparation were determined. In addition, using tandem mass spectrometry, the differential fragmentation patterns between porcine heparin, Lovenox, and Fragmin were determined for all component composition. This method provides an alternate method to examine the structural differences between heparin preparations. A final demonstration of the utility of multiplex tagging examined the N-linked glycans released from a-1-acid glycoprotein from four species. Glycoprotein quantities were normalized to quantitate the abundances of glycans present and the tandem MS of each composition was analyzed.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 对糖组学的兴趣日益增加，导致质谱法广泛用于测定生物系统中表达的碳水化合物组分。 然而，由于仪器和样品的可变性，不同样品中碳水化合物的定量仍然是一项艰巨的任务。 在这里，我们显示cusyom合成的多重稳定同位素标记的标签，具有广泛的适用性，同时定量的四个样品在同一个质谱实验。 探索来自各种来源的多重稳定同位素标记的效用的结果已经被证明。 合成了四种形式（+0、+4、+8、+12）的稳定同位素标记标签，用于标记聚糖的还原端。 硫酸软骨素蛋白聚糖（CS-PG）类的糖胺聚糖（GAG）和药用低分子量（LMWT）肝素使用酶部分解聚形成寡糖分布。 这些寡糖通过还原胺化用四链体稳定的含同位素的标签标记。 合并所得标记的GAG，通过高效尺寸排阻色谱法（Superdex peptide 3.2/30，Beckman Gold 125溶剂模块）分离，并使用Applied Biosystems QSTAR Pulsar-I（Q-ToF）质谱仪以负电喷雾模式进行分析。 此外，还对从四种菌种释放的PNGase F α-1-酸性聚糖进行了多重标记和分析。 将稳定同位素模块并入寡糖结构中的一系列糖组学标签用于标记从CS-PG、LMWT肝素释放的聚糖和来自a-1酸性糖蛋白的N-连接聚糖。 MS模式中的离子丰度用于量化四个样品中的每一个中的给定样品产生的离子。 这些结果进一步证明了使用稳定同位素标签通过碳水化合物的多重分析进行定量的原理。 软骨素酶ABC用于从聚集蛋白聚糖、双糖链蛋白聚糖、软骨提取物和核心蛋白聚糖中去除释放的GAG链。 将CS-蛋白聚糖的浓度标准化，以允许对相对CS量进行质谱分析。 利用各糖型（CSA、CSB和CSC）的独特裂解模式，通过串联质谱分析获得CS链异构体含量的其他信息。 进行额外的实验以证明标签在GAG检查中的更大效用。 低分子量肝素（Lovenox和法安明）用肝素裂解酶I、II和III尽可能解聚。 对裂解酶处理有抗性的肝素（和LMWT肝素）的显著部分应由含有三硫酸化GlcN残基的四糖组成，该残基来源于抗凝血酶III结合所需的五糖序列。 测定每种LMWT肝素制剂的裂解酶抗性结构的量。 此外，使用串联质谱法，测定了所有组分组成的猪肝素、Lovenox和法安明之间的差异裂解模式。 该方法提供了检查肝素制剂之间结构差异的替代方法。 多重标记的效用的最后演示检查了从四个物种的α-1-酸性糖蛋白释放的N-连接聚糖。 将糖蛋白量归一化以定量存在的聚糖的丰度，并分析每种组合物的串联MS。