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

• Published in: Italian journal of animal science Vol. 21; no. 1; pp. 1508 - 1519
• Main Authors: Sechovcová, Hana, Rudl Kulhavá, Lucie, Fliegerová, Kateřina, Killer, Jiří, Kopečný, Jan
• Format: Journal Article
• Language: English
• Published: Bologna Taylor & Francis 31.12.2022; Taylor & Francis Ltd; Taylor & Francis Group
• Subjects: 2DE; Butyrivibrio fibrisolvens; Carbon sources; Fermentation; Fructose-bisphosphate aldolase; Glycolysis; Hemicellulose; label-free; Liquid chromatography; Mass spectroscopy; Membrane proteins; Methods; Nucleotidase; Polysaccharides; Protein biosynthesis; Protein transport; Proteins; Proteomics; Rumen; Xylan
• ISSN: 1828-051X; 1594-4077; 1828-051X
• DOI: 10.1080/1828051X.2022.2129477

The aim of this study was the comparison of label-free method with 2DE to other analytical method of bacterium Butyrivibrio fibrisolvens 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). B. fibrisolvens 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 B. fibrisolvens. HIGHLIGHTS 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. Butyrivibrio fibrisolvens 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.