Proteomics: Edge over other technologies
Proteomics, one of the major tools of ‘omics’ is evolving phenomenally to identify proteins involved in signaling pathways at the end of twentieth century. The term proteomics was coined in 1997 in analogy with genomics, the study of the genome. The word proteome is actually a combination of protein and genome and was coined by Mark Wilkins in 1994. Proteome is the entire set of proteins produced by a living organism and proteomics deals with the study of proteome. Proteomics has advanced over time to enrich the understanding of salt stress mechanism in plants. While the other two fields of ‘omics’ i.e; genomics and transcriptomics deal with the analysis of genes, regulatory elements and their transcripts, the field of proteomics deals with the analyses of proteins (Ashwin et al. 2017). Proteomic based technologies had been widely used in several crop species to understand the changes in the cellular activities at protein level under salt stress.
Proteins are the important macromolecules exhibiting diverse functions in plant stress tolerance. They act as enzymes, exhibit protective functions, interact with other proteins and other biomolecules, and scavenge ROS either directly via chemical reactions or indirectly via metal cofactors. It is therefore highly important to study changes in proteome composition under stress to uncover key proteins involved in mechanisms underlying plant acclimation to stress. However, it is quite evident that the diverse functionality of one protein depends on its sub-cellular localization, PTMs and interacting partners. Therefore, studies of particulate protein, PTMs as well as protein-protein interactions are extremely important. The major focus of proteomics studies in the future would probably shift from a mere identification of differentially expressed proteins to a proper characterization of protein function in plant stress response (Kosová et al. 2013). 2-DE coupled with mass spectrometry is the widely used quantitative proteomics methods. Nonetheless, the defects of low rate of protein detection, low reproducibility and difficult isolation of hydrophobic proteins restricted the full potential of 2-DE in systematic analysis of proteomic changes (Sun et al. 2017). This technical disadvantage of 2-DE gave rise to the gel-free based protein quantitative approach which is also regarded as shot gun proteomics.
34.3 Technical advances in proteomics
There are ideally three critical stages in proteomic approaches viz., sample preparation, gel/column-based protein/peptide separation, and identification of proteins using MS (Ashwin et al. 2017). In case of model systems including human, yeast and bacterial proteomes advanced proteomic technologies are being developed but they may not be directly applied to plant tissues (Timperio et al. 2008). In case of plant tissue the main critical step for proteome analysis is sample preparation because of rigid cell wall, presence of secondary metabolites like phenolic compounds, polysaccharides etc. These secondary metabolites usually cause protein precipitation during the disruption of tissues. Therefore, it is difficult to obtain high quality protein in case of plants. Moreover, roots and fruits have got least amount of protein content and high amount of interfering substances which makes it challenging to extract protein for proteome analysis. There are many techniques of extracting proteins from plant tissues for proteomic studies but phenol and trichloroacetic acid (TCA)-based extraction methods have been found to give superior