Spots with increased abundance are indicated in red. Proteins were visualized using CBB. Second, we analyzed each of the spot variations in 2DE gels obtained with the two methods from three independent replicates Fig 4 , S4 Fig , S1 Table. Particularly, for 2DE gels of maize mesocotyls, , , and colloidal CBB stained spots were detected in three 2DE maps, respectively, with spots in common. Undeniably, there was a substantial variation in abundance among spots in three independent replicates, which is an inherent drawback of common 2DE.
Another two independent experiments were shown in S4 Fig. Right panel: acetone precipitation. Protein was visualized using colloidal CBB. Overall, the former produced good 2DE maps. Obviously, some spots were preferably extractable to the extraction method, but more spots were lost after simple acetone precipitation, especially high-mass spots in acidic regions. Though simple acetone precipitation worked well for some cell materials [ 17 ]. Many previous studies indicated that simple acetone precipitation precludes production of good 2DE maps due to the presence of high levels of interfering substances in plant materials.
A recent research reported that protein loss is believed to be an inevitable consequence of acetone precipitation of proteome extracts [ 27 ]. In addition, it is worthwhile to note that aqueous TCA precipitation can cause severely denatured proteins that are very difficult to dissolve; hence, this method is rarely used in proteomic analysis.
Thus, we did not compare aqueous TCA precipitation with the modified method in the present study. Previously, Wang et al. The combination of TCA and acetone is more effective than either TCA or acetone alone to precipitate proteins [ 8 , 28 ].
It is noted that some proteins were preferentially extracted by the modified or the classical method, but the rationale remains open to question. Recently, we reported a chloroform-assisted phenol extraction method for depletion of abundant storage protein globulin-1 in monocot seeds maize and in dicot soybean and pea seeds [ 29 ]. The modified method was highly efficient in depleting globulin-1, suggesting another application of the modified method in proteomic analysis.
We observed some significant, repeated differences in abundance of several DAPs between the two methods. There were specific DAPs associated with each method in different samples.
However, the reason behind this phenomenon remains unclear. We tried to analyze the hydropathicity, physicochemical property, and subcellular compartments of these DAPs Table 2 ; however, no definite conclusion could be drawn.
Understandably, different extraction methods can produce protein profiles with substantial or subtle differences [ 30 ], but these inherent differences are difficult to explain, as discussed in a previous study [ 31 ].
It is important to note that protein loss is an inevitable consequence of solvent precipitation, even in the modified method, as observed in acetone precipitation of proteome extracts, including bacterial and mammalian cells [ 32 ]. To summarize, the greatest advantages of the modified method are its simplicity and fast. Despite its steps being similar to aqueous TCA precipitation, the modified method circumvents the drawback of aqueous TCA precipitation, namely, TCA-precipitated proteins being difficult to dissolve.
As the modified method precipitates proteins in aqueous extracts, it is expected to be universally applicable for various plant tissues in proteomic analysis.
Shown were two independent experiments. Shown were three independent experiments. Shown are two independent experiments. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. Background Protein extracts obtained from cells or tissues often contain interfering substances, which must be removed for preparing high-quality protein samples [ 1 ].
Material and methods Plant materials Maize Zea mays L. Download: PPT. Fig 1. Acetone precipitation One-step acetone precipitation was performed as described recently [ 17 ]. Fig 2. Evaluation of the modified method First, we made a comprehensive comparison of protein yields and resolution in 2-DE by the modified and classical methods.
Table 1. Comparison of protein yield and spot number in 2DE between the two methods. Fig 3. Comparison of 2DE protein profiles of maize embryo proteins extracted using two methods. Table 2. The identification of the differential extracted proteins in maize using the two methods. Fig 4. Comparison of 2DE profiles of maize mesocotyl proteins extracted using two methods. Supporting information. S1 Fig. S2 Fig. Comparison of 2DE protein profiles of maize root proteins extracted using two methods.
S3 Fig. Comparison of 2DE protein profiles of maize leaf proteins extracted using two methods. S4 Fig. S1 Table. Comparison of spot variations in 2DE gels of maize mesocotyl. S2 Table. Comparison of spot variations in 2DE gels of maize embryo. References 1. Trichloroacetic acid TCA precipitation of proteins. Cold Spring Harb Protoc. Optimizing protein extraction from plant tissues for enhanced proteomics analysis.
J Sep Sci. Front Plant Sci. Total protein extraction with TCA-acetone. Methods Mol. Sample extraction techniques for enhanced proteomic analysis of plant tissues. Protein extraction from plant tissues for 2DE and its application in proteomic analysis.
Proteomics ; — Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis ; 7: 52—4. View Article Google Scholar 8. Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis.
A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues.
The interaction between proteins is stronger than between the protein and the available water molecules which causes the protein aggregation and precipitation.
Some examples are zinc sulphate and ammonium sulphate. This method is widely used for initial fractioning of different proteins, based on their solubility. Salting precipitation is not harmful for proteins so after the experiment, they can be re-dissolved and keep their biological functions.
Organic solvents can be also used. Adding alcohol to the solution reduces the hydration of the protein, by removing water and surrounding proteins which leads to aggregation and precipitation. To preserve their biological functions, it is important to run the experiments at low temperature. Also, check the pH of the solution and the concentration of the protein. Quality policy. Terms and conditions of sale. Certificate ES Quick questions. Toggle Navigation Products Redox Technologies.
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