Protein abundance in multiplexed samples (PAMUS) for quantitation of Trichoderma reesei secretome.

Protein abundance determination across multiple samples proved to be a daunting task and far fewer methods have been successfully devised for this purpose. Despite the technical challenges faced, protein abundance determination over multiple samples is still an area of interest. Herein, we introduce a new method for estimation of protein abundances in multiplexed samples (PAMUS). Protein abundance in the multiplexed sample comprising of the eight complex secretomes by Trichoderma reesei QM6a and Rut C30 grown in four different carbon sources, namely glucose, cellulose, starch, and a mixture of starch and cellulose was determined. For protein abundance in the multiplexed sample, exponentially modified protein abundance index (emPAI) was used. Using the PAMUS method, we estimated the abundance of extracellular lignocellulolytic proteins secreted by two T. reesei strains in response to various carbon sources. The results reveal that cellulose induces biosynthesis of cellulases. PAMUS analysis of the secretomes implicates T. reesei Rut C30 as a hyper cellulolytic strain and further revealed the optimum concentrations of each secreted enzyme during cellulosic substrate utilization. Our study demonstrates the plausible use of the PAMUS method for designing enzyme cocktails for optimum cellulose hydrolysis, and its potential applications in future studies involving other multiplexed biological samples. BIOLOGICAL SIGNIFICANCE: Relative protein quantitation across multiple complex biological samples dominates the field of quantitative proteomics. Protein abundance determination across multiple samples, on the other hand, proves to be a daunting task and far fewer methods have been successfully devised for this purpose. Despite the technical challenges faced, protein abundance determination over multiple samples is still an area of interest as it provides unique information about the biological processes, and physiological states of particular disease, or that of microbes. This study introduces a new method of estimation of protein abundance in multiplexed samples (PAMUS) which is applied to study eight complex secretomes by Trichoderma reesei QM6a and Rut C30 grown in different carbon sources. The expression levels of proteins in the multiplexed eight complex secretome samples were determined by isobaric tags for relative and absolute quantitation (iTRAQ) reagents coupled with exponentially modified protein abundance index (emPAI) to calculate the abundance of each identified protein in eight conditions. This method with microbial secretome as an example facilitated a direct comparison of the abundance of proteins. This PAMUS could be applied for any multiple biological samples, for example, to study human disease to evaluate dynamic expression of proteins during disease progressions.

Quantitative secretomic analysis of Trichoderma reesei strains reveals enzymatic composition for lignocellulosic biomass degradation.

Trichoderma reesei is a mesophilic, filamentous fungus, and it is a major industrial source of cellulases, but its lignocellulolytic protein expressions on lignocellulosic biomass are poorly explored at present. The extracellular proteins secreted by T. reesei QM6a wild-type and hypercellulolytic mutant Rut C30 grown on natural lignocellulosic biomasses were explored using a quantitative proteomic approach with 8-plex high throughput isobaric tags for relative and absolute quantification (iTRAQ) and analyzed by liquid chromatography tandem mass spectrometry. We quantified 230 extracellular proteins, including cellulases, hemicellulases, lignin-degrading enzymes, proteases, protein-translocating transporter, and hypothetical proteins. Quantitative iTRAQ results suggested that the expressions and regulations of these lignocellulolytic proteins in the secretome of T. reesei wild-type and mutant Rut C30 were dependent on both nature and complexity of different lignocellulosic carbon sources. Therefore, we discuss here the essential lignocellulolytic proteins for designing an enzyme mixture for optimal lignocellulosic biomass hydrolysis.

Proteomic analysis of pH and strains dependent protein secretion of Trichoderma reesei.

Bioenergy, particularly biofuel, from lignocellulosic biomass has been considered as one of the most promising renewable and sustainable energies. The industrial productivity and efficiency of microbial lignocellulolytic enzymes for cellulosic biofuel applications are significantly affected by pH of culture condition. This study established and compared hydrolytic protein expression profiles of Trichoderma reesei QM6a, QM9414, RUT C30 and QM9414MG5 strains at different pH in cellulosic culture media. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of secretome of T. reesei cultured from pH 3.0-9.0 revealed significantly higher hydrolytic protein expressions at acidic pH. The Bray-Curtis similarity indices, clustering, and Shannon diversity index elucidated differences in protein secretion at different pHs in individuals and among the strains. This study demonstrated a comparative lignocellulolytic enzyme secretion profile of T. reesei and its mutants at different pHs and provides pH sensitive and resistance enzyme targets for industrial lignocellulose hydrolysis.