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A Rapid and Universal Workflow for Label-Free-Quantitation-Based Proteomic and Phosphoproteomic Studies in Cereals
- Source :
- Current protocolsLiterature Cited. 2(6)
- Publication Year :
- 2022
-
Abstract
- Proteomics and phosphoproteomics are robust tools to analyze dynamics of post-transcriptional processes during growth and development. A variety of experimental methods and workflows have been published, but most of them were developed for model plants and have not been adapted to high-throughput platforms. Here, we describe an experimental workflow for proteome and phosphoproteome studies tailored to cereal crop tissues. The workflow consists of two parallel parts that are suitable for analyzing protein/phosphoprotein from total proteins and the microsomal membrane fraction. We present phosphoproteomic data regarding quantification coverage and analytical reproducibility for example preparations from maize root and shoot, wheat leaf, and a microsomal protein preparation from maize leaf. To enable users to adjust for tissue specific requirements, we provide two different methods of protein clean-up: traditional ethanol precipitation (PC) and a recently developed technology termed single-pot, solid-phase-enhanced sample preparation (SP3). Both the PC and SP3 methods are effective in the removal of unwanted substances in total protein crude extracts. In addition, two different methods of phosphopeptide enrichment are presented: a TiO
- Subjects :
- Phosphopeptides
Proteomics
General Immunology and Microbiology
Ethanol
Proteome
General Neuroscience
Reproducibility of Results
Health Informatics
Ferric Compounds
General Biochemistry, Genetics and Molecular Biology
Workflow
Medical Laboratory Technology
Tandem Mass Spectrometry
General Pharmacology, Toxicology and Pharmaceutics
Phosphorylation
Edible Grain
Chromatography, Liquid
Subjects
Details
- ISSN :
- 26911299
- Volume :
- 2
- Issue :
- 6
- Database :
- OpenAIRE
- Journal :
- Current protocolsLiterature Cited
- Accession number :
- edsair.doi.dedup.....2b91033c5c7b7afeaeea81dc315ddfaa