EQUILIBRIUM SEC MASS SPECTROMETRY FOR STUDY OF CARBOHYDRATE-PROTEIN BINDING

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。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。所列机构为中心机构，不一定为研究者机构。碳水化合物通过与不同的细胞外基质蛋白相互作用在介导生物过程中发挥重要作用。为了理解这些相互作用的生理意义，有必要分析碳水化合物结构和结合之间的关系。质谱已被证明是研究碳水化合物结构的有力方法。用于测量碳水化合物-蛋白质结合亲和力的平衡尺寸排阻色谱（SEC）与MS的组合克服了传统限制，即需要纯化寡糖。肝素，硫酸乙酰肝素家族的糖胺聚糖（GAG）的最高度硫酸化的成员，和生物相关的肽之间的结合相互作用进行了研究，在这份报告中。 使用0.1 mL/min的流速，用含有0.1 M乙酸铵（pH 7）和已知浓度肝素的缓冲液平衡Superdex肽（3.2 mm x 30 cm）SEC柱（来自阿默舍姆Pharmacia）。将肽溶解在移动的相缓冲液中，然后将10 μ L该溶液注入SEC柱。使用Hewlett Packard 1090系列HPLC在214 nm处连续监测流出溶液。使用仅含缓冲液的移动的流动相和含肝素的流动相分析五种感兴趣的肽。在第二系列实验中，测试了较小直径的SEC柱（1 mm × 30 cm）和生理缓冲液（0.1M Tris，0.15M NaCl，pH 7）。 使用平衡SEC，可以通过以下等式计算碳水化合物-蛋白质结合亲和力：A=（E0-E）/E0，A=A？/（1+Kd/[肝素]），其中E =洗脱体积，A是在移动的相中存在肝素的情况下观察到洗脱体积降低的因子。一个？表示A的最大值（当肝素浓度饱和时）。在洗脱肽上获得质谱允许分析复杂混合物，并鉴定在移动的相中结合肝素的组分。 在这些实验中使用的五种肽是：纤连蛋白肽FN-C/H II（KNNQKSEPLIGRKKT）和FN-C/H V（WQPPRARI）、P物质、胃泌素和水蛭素。已知纤连蛋白肽结合肝素，并且是阳性对照。P物质肽是一种基本的神经肽，测定其与肝素的结合以检验这种结合是其生物活性的重要机制的假设是有意义的。最后两种肽是酸性的，用作阴性对照。使用分别用8 pmol/L和80 pmol/L肝素浓度平衡的SEC柱进行一系列肽注射。结果表明，随着肝素浓度的增加，FN-C/H II以显著更低的体积洗脱。该肽的结合不同于FN-C/HV，因为它需要较低浓度的肝素来改变洗脱体积。使用该系统，P物质清楚地显示出结合肝素，这是具有相当大的生物学意义的结果。正如预期，胃泌素和水蛭素的洗脱体积作为移动的相中肝素浓度的函数保持不变。这些结果表明，平衡SEC色谱法是鉴定肽和肝之间生物学相关结合相互作用的有用手段。

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. Carbohydrates play major roles in mediating biological processes through their interactions with different extracellular matrix proteins. To understand the physiological significance of these interactions, it is necessary that relationships between carbohydrate structure and binding be analyzed. Mass spectrometry has been demonstrated to be a powerful method to study the structures of carbohydrates. The combination of equilibrium size exclusion chromatography (SEC) with MS for measuring carbohydrate-protein binding affinities overcomes traditional limitations, i.e. the need to purify oligosaccharides. Binding interactions between heparin, the most highly sulfated member of the heparan sulfate family of glycosaminoglycans (GAGs), and biologically relevant peptides has been studied in this report. A Superdex peptide (3.2mm x 30cm) SEC column (from Amersham Pharmacia) was equilibrated with buffer containing 0.1 M ammonium acetate (pH 7) and a known concentration of heparin using a flow rate of 0.1 mL/min. Peptides were dissolved in mobile phase buffer and 10 ¿L of this solution was then injected onto the SEC column. The effluent solution was continuously monitored at 214nm using a Hewlett Packard 1090 Series HPLC. Five peptides of interests were analyzed using mobile phases containing buffer only and those containing heparin. In a second series of experiments a smaller diameter SEC column (1mm x 30cm) and a physiological buffer (0.1 M Tris, 0.15 M NaCl, pH 7) were tested. Using equilibrium SEC, the carbohydrate-protein binding affinity can be calculated by the following equation: A=(E0-E)/E0, A=A?/(1+Kd/[heparin]), where E = the elution volume and A is the factor whereby the elution volume is observed to decrease in the presence of heparin in the mobile phase. A? is taken to represent the maximum value of A (when the heparin concentration is saturating). Acquiring mass spectra on the eluting peptides allows analysis of complex mixtures with identification of components that bind heparin in the mobile phase. The five peptides used in these experiments are: fibronectin peptides FN-C/H II, (KNNQKSEPLIGRKKT) and FN-C/H V (WQPPRARI), substance P, gastrin and hirudin. The fibronectin peptides are known to bind heparin and are positive controls. Substance P peptide is a basic neuropeptide, and it was of interest to determine its binding to heparin to test the hypothesis that such binding is an important mechanism in its biological activity. The last two peptides are acidic and serve as negative controls. A series of peptide injections were made using the SEC column equilibrated with heparin concentrations of 8pmol/¿L and 80pmol/¿L, respectively. The results demonstrate that FN-C/H II eluted with a significantly lower volume with increasing the concentration of heparin. The binding of this peptide was distinct from FN-C/H V in that it required a lower concentration of heparin to shift the elution volume. Using this system, substance P is clearly shown to bind heparin, a result that is of considerable biological interest. As expected, the elution volumes of gastrin and hirudin remained unchanged as a function of heparin concentration in the mobile phase. These results demonstrate that equilibrium SEC chromatography is a useful means for identifying biologically relevant binding interactions between peptides and hepar