Advantages of label free method in comparison with 2DE proteomic analysis of <i>Butyrivibrio fibrisolvens</i> 3071 grown on different carbon sources

The aim of this study was the comparison of label-free method with 2DE to other analytical method of bacterium <i>Butyrivibrio fibrisolvens</i> extracellular protein samples. Label-free quantification (LF) method performed in this work was compared with previously obtained results of proteomic analysis using two-dimensional electrophoresis (2DE) and nano-liquid chromatography coupled with mass spectroscopy (nLC/MS). <i>B. fibrisolvens</i> as an important plant fibre degrader was cultivated on four different carbon sources (including xylan, xylose, glucose, and a mixture of xylan with glucose). The impact of growth substrate on protein profile was assessed by six pair-wise comparisons evaluating the significantly differently abundant extracellular proteins. Gel-free and gel-based methods resulted in substantially dissimilar results. The LC-MS/MS approach detected substrate-dependent differences in transport and binding membrane proteins (TBP) and nucleotidase, while the 2DE approach detected substrate effect on proteins included in protein synthesis and butyrate synthesis. On the other hand, both methods observed differentially regulated proteins involved in the glycolytic pathway, however, the only shared enzyme (protein detected both by 2DE and LC-MS/MS approach) was fructose-bisphosphate aldolase. The LC-MS/MS approach detected a high abundance of separated peptides. However, it cannot be easily considered as supreme to 2DE analysis. Both methods differ in sample preparation, their advantages and limitations. The study findings indicate these methods can complement each other and together they elucidate better the metabolic functions of <i>B. fibrisolvens</i>.HIGHLIGHTSComparison of LF and 2DE analysis showed limitations connected with a narrow optimised pH interval and number of affected proteins in 2DE in contrary to LF results.<i>Butyrivibrio fibrisolvens</i> culture showed the same proteomic changes in the presence of different saccharides as the whole rumen fluid in vivo.Selected proteins could be used for the monitoring of polysaccharides fermentation in the rumen and be used for optimisation hemicellulose degradation and overall feed utilisation. Comparison of LF and 2DE analysis showed limitations connected with a narrow optimised pH interval and number of affected proteins in 2DE in contrary to LF results. <i>Butyrivibrio fibrisolvens</i> culture showed the same proteomic changes in the presence of different saccharides as the whole rumen fluid in vivo. Selected proteins could be used for the monitoring of polysaccharides fermentation in the rumen and be used for optimisation hemicellulose degradation and overall feed utilisation.

本研究旨在针对溶纤维丁酸弧菌（Butyrivibrio fibrisolvens）的胞外蛋白样品，对比无标记定量（label-free quantification, LF）法、二维凝胶电泳（two-dimensional electrophoresis, 2DE）与其他分析方法的应用效果。本研究采用的无标记定量方法，与此前采用二维凝胶电泳及纳米液相色谱-质谱联用（nano-liquid chromatography coupled with mass spectroscopy, nLC/MS）开展的蛋白质组学分析结果进行了对比。

作为重要的植物纤维降解菌，溶纤维丁酸弧菌在四种不同碳源（木聚糖、木糖、葡萄糖及木聚糖与葡萄糖的混合物）中完成培养。通过六组两两比对，对胞外蛋白丰度存在显著差异的样本进行评估，以此分析生长底物对蛋白谱的影响。

无凝胶法与基于凝胶的方法所得结果存在显著差异。液相色谱-串联质谱（LC-MS/MS）方法可检测到转运与结合膜蛋白（transport and binding membrane proteins, TBP）及核苷酸酶的底物依赖性差异，而二维凝胶电泳方法则可检测到底物对蛋白质合成及丁酸合成相关蛋白的调控作用。另一方面，两种方法均观测到糖酵解通路中存在差异调控的蛋白，但仅果糖-二磷酸醛缩酶这一种酶（蛋白）同时被2DE与LC-MS/MS方法检测到。

液相色谱-串联质谱方法可检测到大量分离得到的肽段，但不能简单认定其优于二维凝胶电泳分析。两种方法在样品制备流程、优势与局限性方面均存在差异。本研究结果显示，两种方法可互为补充，共同更全面地阐明溶纤维丁酸弧菌的代谢功能。

## 研究亮点
1. 无标记定量与二维凝胶电泳分析的对比结果显示，相较于无标记定量方法，二维凝胶电泳存在优化pH区间狭窄、受影响蛋白数量有限的局限性。
2. 溶纤维丁酸弧菌在不同糖类环境下的蛋白质组变化，与体内全瘤胃液的蛋白质组变化一致。
3. 筛选得到的目标蛋白可用于瘤胃内多糖发酵的监测，亦可为半纤维素降解优化及整体饲料利用率提升提供参考。

搜集汇总

背景与挑战

背景概述

该数据集基于一项蛋白质组学研究，比较了无标记方法（label-free）与二维电泳（2DE）在分析Butyrivibrio fibrisolvens 3071细菌在不同碳源培养下的细胞外蛋白质样本时的表现，发现两种方法在检测蛋白质差异上各有侧重，可以互补使用以更好地理解细菌代谢功能。数据集包含相关实验数据，发布于2022年，涉及生物物理学和微生物学等领域。