Method for Label-Free Quantitative Proteomics for Sorghum bicolor L. Moench
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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.
KeywordsAbiotic stress molecular method quantitative proteomics sorghum total protein extraction
2-Dimensional Gel Electrophoresis
- 2-D DIGE
2-Dimensional Difference Gel Electrophoresis
Matrix Assisted Laser Desorption/Ionization – Time of Flight - Mass Spectrometry
Coomassie Brilliant Blue Stain
2-Dimensional Liquid Chromatography Mass Spectrometry
Days After Germination
Sodium Dodecyl Sulphate
Poly Vinyl Poly Pyrrolidone
- 1-D SDSPAGE
1-Dimensional SDS Polyacrylamide Gel Electrophoresis
Ethylene Diamine Tetra Acetic Acid
Protein Lynx Global Server
Isobaric Tags for Relative and Absolute Quantitation
The authors would like to acknowledge the financial, infrastructural and academic support provided by RPDC (DBT-IUSSTF JCERDC project), Legumes Pathology and AGL at ICRISAT, India, Departmental Research and Developmental Program (DRDP), IBB, Savitribai Phule Pune University (SPPU), Pune, India, and Birla Institute of Technology, Mesra, India towards completion of research work. We also deeply appreciate and acknowledge DST-SERB, India. The authors also acknowledge Mr. Arun Surendran and the technical team at Proteomic Core Facility, Rajiv Gandhi Centre for Biotechnology (RGCB), India for supporting the work.
AAS conducted all experimental work at ICRISAT, India, quality control on proteomics analysis samples, preparation of raw proteomic data for bioinformatic analysis, has written major portion of the manuscript and formatting of the same. ANN conducted the experimental work at IBB Pune and a part of bioinformatics analysis on proteomic data and written relevant sections of the manuscript arising from this work. AJ and the technical team at RCGB conducted all LC-MS/MS based proteomic analysis, generated the raw differential proteomic data across the different sorghum cultivars in the study and written relevant sections of the manuscript arising from this work. VAT and SPR have planned, designed and supervised the experimental and bioinformatics works, as principal investigators at IBB, SPPU, Pune and ICRISAT, Patancheru respectively. They acquired funding for the work and edited and reviewed the manuscript for submission. All authors read and approved the final manuscript.
The major funding for this work was provided by the Research Program on Dry Cereals (RPDC) at ICRISAT, India under the Department Of Biotechnology (DBT – Government of India) – Indo-US Science and Technology Forum (IUSSTF) Joint Clean Energy Research And Development Centre (JCERDC) project. This covered financial support to primary author for conducting the research work at ICRISAT; material, equipment and reagent expenses; accessing infrastructural facilities and experimental space in the Department of Legumes Pathology and Applied Genomics Laboratory at ICRISAT, India as well as proteomics work done at the Rajiv Gandhi Centre for Biotechnology (RCGB), Kerala, India. Financial support for the experimental work and bioinformatics analysis done at Institute of Bioinformatics and Biotechnology (IBB) Pune, India was through research grant from the Department of Science and Technology (DST) – Science and Engineering Research Board (SERB), Government of India.
Compliance with Ethical Standards
Ethics Approval and Consent to Participate
Consent for Publication
Conflict of Interest
The authors declare that they have no conflict of interest.
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