Development of a Multipoint Quantitation Method to Simultaneously Measure Enzymatic and Structural Components of the Clostridium thermocellum Cellulosome Protein Complex

Clostridium thermocellum has emerged as a leading bioenergy-relevant microbe due to its ability to solubilize cellulose into carbohydrates, mediated by multicomponent membrane-attached complexes termed cellulosomes. To probe microbial cellulose utilization rates, it is desirable to be able to measure the concentrations of saccharolytic enzymes and estimate the total amount of cellulosome present on a mass basis. Current cellulase determination methodologies involve labor-intensive purification procedures and only allow for indirect determination of abundance. We have developed a method using multiple reaction monitoring (MRM-MS) to simultaneously quantitate both enzymatic and structural components of the cellulosome protein complex in samples ranging in complexity from purified cellulosomes to whole cell lysates, as an alternative to a previously developed enzyme-linked immunosorbent assay (ELISA) method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision for determination of the mass concentration of cellulosome components.

热纤梭菌（Clostridium thermocellum）因可通过被称为纤维小体（cellulosome）的多组分膜附着复合物介导，将纤维素降解为糖类，已成为生物能源领域极具代表性的模式微生物。为探究微生物的纤维素利用速率，亟需实现糖化酶浓度的定量检测，并以质量为基准估算样品中纤维小体的总含量。当前的纤维素酶定量方法需依赖耗时费力的纯化流程，且仅能间接测定其丰度。本研究开发了一种基于多反应监测质谱（multiple reaction monitoring，MRM-MS）的分析方法，可同时定量复杂度跨度从纯化纤维小体到全细胞裂解液的样品中纤维小体蛋白复合物的酶学组分与结构组分，作为此前开发的纤维小体酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）定量方法的替代方案。所有样品的技术重复实验中，纤维小体质量浓度的相对标准偏差均优于5%，表明该方法用于纤维小体组分质量浓度测定时具有较高的精密度。