Surroca Y, Haverkamp J, Heck A J R
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands.
J Chromatogr A. 2002 Sep 13;970(1-2):275-85. doi: 10.1016/s0021-9673(02)00884-1.
Heat-induced aggregation of bovine beta-lactoglobulin AB (10 mg/ml) was studied at 68.5 degrees C at two different pH values (6.7, 4.9) using gel electrophoresis techniques and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis under non-reducing and reducing conditions showed that in the early stages of the aggregation of beta-lactoglobulin disulfide linked aggregates were formed on heating at pH 6.7, but not at pH 4.9. We related this result to the pH-dependent activity of the free thiol group at C121. Mass spectrometric analyses were conducted in two steps. The first involved the analysis of intact non-native monomers and dimers following their ultrasonic passive elution into a suitable solvent mixture in order to confirm the identity of the different gel bands. The second step comprises the analysis of in-gel digests for the determination of disulfide patterns in non-native monomers, covalent dimers and trimers. The results of in-gel digestions analyzed by mass spectrometry suggest that non-native dimers could result from the formation of inter-molecular disulfide bonds C121-C66, C160-C160, or C121-C160. Moreover, two inter-molecular bonds C121-C66 and C160-C160 between two and the same monomer units have been detected, which may play an important role in limiting the process of covalent beta-lactoglobulin network formation. The combination of SDS-PAGE and MALDI-TOF MS enables us to understand the mechanism of beta-lactoglobulin aggregation at the macromolecular level.
利用凝胶电泳技术和基质辅助激光解吸电离质谱（MALDI-TOF MS），在68.5摄氏度、两种不同pH值（6.7、4.9）条件下研究了热诱导牛β-乳球蛋白AB（10毫克/毫升）的聚集情况。在非还原和还原条件下的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，在β-乳球蛋白聚集的早期阶段，在pH 6.7加热时会形成二硫键连接的聚集体，而在pH 4.9时则不会。我们将这一结果与C121处游离巯基的pH依赖性活性相关联。质谱分析分两步进行。第一步是在将完整的非天然单体和二聚体超声被动洗脱到合适的溶剂混合物后，对其进行分析，以确认不同凝胶条带的身份。第二步包括对凝胶内消化产物进行分析，以确定非天然单体、共价二聚体和三聚体中的二硫键模式。通过质谱分析凝胶内消化产物的结果表明，非天然二聚体可能是由分子间二硫键C121-C66、C160-C160或C121-C160的形成导致的。此外，还检测到两个相同单体单元之间的两个分子间键C121-C66和C160-C160，这可能在限制共价β-乳球蛋白网络形成过程中起重要作用。SDS-PAGE和MALDI-TOF MS的结合使我们能够在大分子水平上理解β-乳球蛋白聚集的机制。
