EQUILIBRIUM SEC MASS SPECTROMETRY FOR STUDY OF CARBOHYDRATE-PROTEIN BINDING

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。碳水化合物通过与不同的细胞外基质蛋白相互作用，在调节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。质谱仪已被证明是研究碳水化合物结构的一种强有力的方法。平衡尺寸排阻色谱(SEC)与MS相结合来测量碳水化合物与蛋白质的结合亲和力，克服了传统的限制，即需要提纯低聚糖。本文研究了糖胺多聚糖(GAG)硫酸乙酰肝素家族中硫酸化程度最高的成员肝素与生物相关多肽之间的结合作用。Superdex多肽(3.2 mm×30 cm)SEC柱(来自amersham Pharmacia)用含有0.1M醋酸铵(PH 7)和已知浓度的肝素的缓冲液平衡，流速为0.1mL/min。在流动相缓冲液中溶解多肽，然后将10%的L溶液进样到Sec柱上。使用Hewlett Packard 1090系列高效液相色谱法在214 nm处连续监测流出液。用只含缓冲液的流动相和含肝素的流动相分析了5个感兴趣的多肽。在第二系列实验中，测试了较小直径的SEC柱(1 Mm X 30 Cm)和生理缓冲液(0.1M Tris，0.15M氯化钠，pH 7)。利用平衡Sec，碳水化合物-蛋白质结合亲和力可由下列方程计算：A=(E0-E)/E0，A=A？/(1+Kd/[肝素])，其中E=洗脱体积，A是在流动相中有肝素存在时观察到洗脱体积减小的因子。一个?表示A的最大值(当肝素浓度饱和时)。通过获取洗脱肽的质谱图，可以分析复杂混合物，并鉴定流动相中与肝素结合的成分。这些实验中使用的五个肽是：纤维连接蛋白多肽FN-C/H II，(KNNQKSEPLIGRKKT)和FN-C/H V(WQPPRARI)，P物质，胃泌素和水飞蓟素。纤维连接蛋白多肽已知能与肝素结合，是阳性对照。P物质多肽是一种基本的神经肽，通过测定其与肝素的结合来验证其与肝素结合是其生物活性的重要机制这一假说。最后两个多肽是酸性的，作为阴性对照。用肝素浓度分别为8pmol/℃的L和80pmol/℃的L平衡的Sec色谱柱进行了一系列的多肽注射。结果表明，随着肝素浓度的增加，FN-C/HⅡ的洗脱体积明显减小。与FN-C/H-V的结合不同于FN-C/H-V，因为它需要较低浓度的肝素来改变洗脱体积。利用这个系统，P物质被清楚地显示为与肝素结合，这一结果具有相当大的生物学意义。正如预期的那样，胃泌素和水飞蓟素的洗脱体积与流动相中肝素浓度的函数保持不变。这些结果表明，平衡SEC层析是鉴定多肽与肝脏之间具有生物相关性的结合作用的有效手段

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