Abstract
In more than 20 years of the proteomics era, considerable technical developments and scientific discoveries have contributed to the advancement of this field of research. Gel-based proteomics has been popular for studying the proteomic changes during growth and development of plants as well as for the analysis of responses to different biotic and abiotic stimuli. The most widely used gel-based technique is two dimensional (2D) gel electrophoresis, in which around 2000 protein spots can be clearly visualized and processed prior to identification by mass spectrometry. Although many techniques have been developed to improve the quality and number of spots in a 2D gel, these enhancements are still not good enough to study the entire cellular proteome. It is important to also consider low abundant proteins, many of which could play critical roles in particular biological processes. Considering that thousands of proteins are expressed at a given time and each protein can undergo one or more post-translational modifications—are we able to capture all this information and the entire proteome using gel-based proteomic techniques? Are gel-free alternatives capable of addressing these challenges? The answer is no for both approaches. So, how long are we going to run traditional 1D and 2D gels in our research? Though we have been successful in developing many advanced gel-free proteomic techniques to study the cellular proteome, we still lack advances that enable the examination of each and every protein expressed in a system as well as any post-translational modification (PTM) at a given time. Gel-free and gel-based techniques may complement each other, however there needs to be synergy or a “technical fusion” in which researchers can find a balance between quick and efficient methods for studying whole cell proteomes in plants.
Highlights
Considering that thousands of proteins are expressed at a given time and each protein can undergo one or more post-translational modifications—are we able to capture all this information and the entire proteome using gel-based proteomic techniques? Are gel-free alternatives capable of addressing these challenges? The answer is no for both approaches
How long are we going to run traditional 1D and 2D gels in our research? Though we have been successful in developing many advanced gel-free proteomic techniques to study the cellular proteome, we still lack advances that enable the examination of each and every protein expressed in a system as well as any post-translational modification (PTM) at a given time
Different proteomic tools have been developed in the past few decades, most of which were first used for studying animal proteomes and later adopted for plants
Summary
Many techniques have been developed to improve the quality and number of spots in a 2D gel, these enhancements are still not good enough to study the entire cellular proteome. This article covers some of the more recent studies in the MS workflow before raising the question of how can we improve the output by merging different techniques i.e., development of different methods in gel-based and gel-free techniques to improve protein separation, digestion and recovery of peptides, and MS or MS/MS data analysis.
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