Method for Label-Free Quantitative Proteomics for Sorghum bicolor L. Moench

Abstract

Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.

Label-free quantitative proteomics of Sorghum bicolor reveals the proteins strengthening plant defense against insect pest Chilo partellus

“…Total protein extraction was done using a phenol extraction method as described earlier [ 34 ]. In short, S. bicolor leaf tissues stored at − 80 °C were ground to a fine powder in liquid nitrogen with mortar and pestle.…”

“…Proteins were reconstituted to a final concentration of 1 μg/μL with 0.1% Rapigest™ in 50 mM ammonium bicarbonate. One hundred microgram of protein from each sample (1 μg/μL) was used for in-solution reduction and alkylation followed by trypsin digestion to obtain the peptides [ 34 ].…”

“…The instrument was operated and controlled by MassLynx4.1 SCN781 software. The peptide resolution conditions were as detailed by Sharan et al [ 34 ]. SYNAPT® G2 High Definition MS™ System (HDMS E System) (Waters Corporation, Milford, USA) was used to carry out mass spectrometry analysis of eluting peptides with instrument settings as; nano-ESI capillary voltage – 3.4 kV, sample cone - 40 V, extraction cone - 4 V, IMS gas (N 2 ) flow - 90 (ml/min).…”

“…SYNAPT® G2 High Definition MS™ System (HDMS E System) (Waters Corporation, Milford, USA) was used to carry out mass spectrometry analysis of eluting peptides with instrument settings as; nano-ESI capillary voltage – 3.4 kV, sample cone - 40 V, extraction cone - 4 V, IMS gas (N 2 ) flow - 90 (ml/min). All analyses were performed using positive mode ESI using a NanoLockSpray™ source as detailed in [ 34 ]. Protein identification and label-free relative protein quantification were done by analyzing LC-MS/MS data using ProteinLynx Global Server™ v2.5.3 (PLGS, Waters Corporation) for each technical replicate.…”

Potential food applications of sorghum (Sorghum bicolor) and rapid screening methods of nutritional traits by spectroscopic platforms

“…Commercial agriculture has four primary sectors; ethanol production (Cole et al, 2017), animal feedstock (Ronda et al, 2019), as a cover crop (Mesbah et al, 2019), and food product formulations (Rashwan et al, 2021). The ability to grow in high-salinity conditions, tolerate biotic and abiotic stresses (Sharan et al, 2018), and become dormant in adverse environmental conditions (Labuschagne, 2018) make sorghum an ideal crop to address global food security. When the environmental conditions become favorable, sorghum seeds at the dormant stage start to germinate, and it is a competitive advantage over other commercial crops.…”