ANALYSIS OF NITROUS ACID DEPOLYMERIZATION PRODUCTS OF GLYCOSAMINOGLYCANS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 前言：硫酸乙酰肝素(HS)糖胺多聚糖具有多种生物学功能。为了更好地了解它们的生物学作用，有必要了解HS的结构。已经开发了涉及酶消化的方法来确定硫酸盐/乙酰化修饰的类型和位置。然而，生成的双糖在非还原端具有不饱和性，从而消除了己糖醛酸原来的表观性质。肼裂解和亚硝酸处理是一种经典的GAG解聚方法，保留了糖醛酸的异构化。到目前为止，它还没有与质谱学检测相结合。本工作结合LC-MS和LC-MS/MS对HONO生成的双糖进行分析，以获得有关硫酸盐化模式和糖醛酸异构化的信息。 方法：用70%肼(含1%硫酸肼)对15 g完整肝素和HS进行脱乙酰化。混合物被加热、冷却、蒸发至干燥，然后脱盐。对于母鸡，加入pH为4的HONO试剂，15分钟后将pH调节到8.5。对于HS，加入冷的pH 1.5 HONO试剂，10分钟后将pH调节到4，然后在pH 4的亚硝酸(HONO)处理。肝素(Hep)样品在pH 1.5的HONO处理。所得二糖用NaBH4还原，然后用尺寸排阻色谱(SEC)、LC-MS和LC-MS/MS分析。 初步资料：在大多数研究中，HS的双糖分析是通过肝素裂解酶消化的方法进行的。裂解酶催化的裂解在非还原末端产生-4，5-六碳残基，导致糖醛酸的C5碳平坦化。一种替代方法是使用肼分解和亚硝酸(HONO)在pH为1.5和4的条件下处理，以生成由含醛的糖醛酸(idOA或GLCA)和2，5-脱水甘露糖(AnMan)组成的双糖单元。 第一组实验包括确定肼分解和亚硝酸处理的最佳条件。测试了不同联氨溶液的用量和加热时间。我们发现，在96℃下，用20°L的肼溶液作用4小时，可得到几乎完全的脱乙酰基。在HONO处理后，用0.5M的NaBH4在37℃下还原1h，然后进行SEC-MS分析。化学法得到的猪肠道粘膜HEP、HS和E.ColiK5细菌培养物中Hen的双糖组成与酶法制备的HEP和Hs的双糖组成相似，说明肼分解和HONO处理提供了有关双糖组成的可靠信息。 我们还发现，Hen和Hep分别至少含有95%的Glca和70%的idoA。从这两个样品中获得的双糖的串联MS产生了跨环裂解以及硫酸盐基团的损失。然而，串联MS谱中观察到的差异是由于硫酸盐位置而不是己糖醛酸的同一性。这些结果证明了LC/MS分析HONO生成的GAG双糖的有效性。很可能在LC/MS系统中使用更高分辨率的层析模式将允许直接分离双糖同分异构体以便于其鉴定。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Introduction: Heparan sulfate (HS) glycosaminoglycans have been implicated in a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS. Methods involving enzymatic digestion have been developed to determine the type and location of sulfation/acetylation modifications. However, the generated disaccharides have ¿-unsaturation at the non-reducing end, thus, erasing the original epimeric nature of the hexuronic acid. Hydrazinolysis and nitrous acid treatment is a classic method for GAG depolymerization that retains uronic acid epimerization. To date, it has not been combined with mass spectrometric detection. This work shows combined LC-MS and LC-MS/MS for analysis of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Methods: 15 ¿g of intact heparosan (Hen) and HS were deacetylated by adding 70% hydrazine containing 1% hydrazine sulfate. The mixture was heated, cooled, evaporated to dryness, and desalted. For the Hen, pH 4 HONO reagent was added and pH was adjusted to 8.5 after 15 minutes. For the HS, cold pH 1.5 HONO reagent was added, pH adjusted to 4 after 10 minutes, followed by nitrous acid (HONO) treatment at pH 4. The heparin (Hep) sample was subjected to HONO treatment at pH 1.5. The resulting disaccharides were reduced with NaBH4 and then analyzed using size exclusion chromatography (SEC) LC-MS and LC-MS/MS. Preliminary Data: In most studies, disaccharide analysis of HS was performed by means of enzymatic digestion with heparin lyases. The lyase catalyzed cleavage generates a ¿-4,5-HexA residue at the nonreducing terminus, resulting in planarization of the C5 carbon of the uronic acid. An alternative is the use of hydrazinolysis with nitrous acid (HONO) treatment at pH 1.5 and 4 to generate disaccharide units composed of uronic acid (IdoA or GlcA) and 2,5-anhydromannose (AnMan) bearing an aldehyde group. The first set of experiments involved determining the optimum conditions for hydrazinolysis and nitrous acid treatment. Various amounts of hydrazine solution and heating times were tested. We found that hydrazinolysis with 20 ¿L of hydrazine solution at 96 oC for 4 hours yielded almost complete deacetylation. After HONO treatment, the disaccharides were reduced with 0.5 M NaBH4 for 1 hour at 37 oC followed by SEC-MS analysis. The compositions of the disaccharides of Hep and HS from porcine intestinal mucosa, and Hen from E.coli K5 bacterial culture obtained using the chemical method were found to be comparable to those obtained by the enzymatic method, showing that the hydrazinolysis and HONO treatment provide reliable information about disaccharide composition. We also found that Hen and Hep contain at least 95% GlcA and 70% IdoA, respectively. Tandem MS of disaccharides obtained from these two samples generated cross-ring cleavages as well as loss of sulfate groups. However, the differences observed in the tandem MS spectra were due to the sulfate position rather than the identity of the hexuronic acid. These results demonstrate the efficacy of LC/MS for analysis of HONO generated GAG disaccharides. It is likely that use of higher resolution chromatography mode with the LC/MS system will allow direct separation of disaccharide epimers to facilitate their identification.