Abstract
The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.
Highlights
To date, a number of different protein extraction methods and solubilization buffers have been applied in plant proteomics studies and they differ from those typically used in prokaryote and animal studies [1]
The 2DE analysis revealed substantial qualitative differences among the five buffer systems tested for the single solubilization process, which can be attributed to their ability to solubilize proteins
The CHAPS-containing buffer system allowed for the visualization of a greater number of protein spots, with >1200 spots resolved on 2DE gels on average (Table 1)
Summary
A number of different protein extraction methods and solubilization buffers have been applied in plant proteomics studies and they differ from those typically used in prokaryote and animal studies [1]. Proteins from apple (Malus domestica), avocado (Persea americana), banana (Musa americana) and orange (Citrus × sinensis) fruits extracted in a study using either a phenol/chloroform or a trichloroacetic acid (TCA) in acetone protocol yielded different proteome profiles [3,4]. The alternative gel-free approaches including protein antibody arrays [10] and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) [11] can overcome some of the limitations of 2DE
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