Abstract
Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK.
Highlights
One of the critical steps in a proteomics study is the selection of the most appropriate protein extraction method
Each one with an equivalent of 230 mg of fresh weight (FW), corresponding to 30 mg of dry powder, were prepared and used to extract protein in triplicate, using five different methods. These methods were: direct extraction in Lysis Buffer (L); direct extraction in Lysis Buffer followed by Cleanup using a commercially available kit (LC); TCA/Acetone Precipitation (T); Phenol Extraction (P); and TCA/Acetone Precipitation followed by Phenol Extraction (TP)
The methods tested were: direct extraction in Lysis Buffer (L); L followed by Cleanup using a commercial kit (LC); TCA/ Acetone Precipitation (T); Phenol Extraction (P); and TCA/Acetone Precipitation followed by Phenol Extraction (TP)
Summary
One of the critical steps in a proteomics study is the selection of the most appropriate protein extraction method. For differential proteomic analysis and protein isoform characterization, it is crucial that the protein extraction method allows the preservation of protein post-translational modifications (PTMs). The protein extraction in a urea-based denaturing buffer can promote protease inhibition without any need for protein precipitation [2]. This type of extraction is, generally considered unsuitable for plant material and it is rarely used in plant proteomics, even though some plant materials (e.g.: plantlets and young leaves) are easy to extract. The authors used the 5th maize leaf as the youngest tissue and concluded that the combination of TCA/acetone precipitation with a phenol extraction was an all-purpose sample preparation method, phosphoproteome conservation was not analyzed
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