As mentioned in our earlier posts, extracting, separating and identifying proteins from plant samples present a unique challenge since they contain significant amounts of proteases and other compounds that may interfere with the process. Generally, there are three critical aspects for successful protein separation:
- Tissue disruption
- The removal of secondary metabolites
- Protein solubilization.
Keep in mind that the sample preparation step determines the success of your experiment so you definitely need to get this right.
Extracting total proteins from recalcitrant plant tissues also requires special considerations. Ideally, you should be able to reproducibly capture all the protein species in a proteome containing the least amount of contaminants. In addition, the extraction process should be properly optimized for the specific plant species, tissue, or cell compartment that you are using. In recent times, several protein extraction protocols such as TCA/acetone extraction, phenol-based extraction and TCA/acetone precipitation/phenol extraction have been developed to provide enhanced plant proteomic analysis.
Specific Considerations for Extracting Proteins from Specific Plant Tissues
Leaves
Using developed or aged leaves for 2-DE analysis can be more difficult than using young leaves since their vacuoles contain higher levels of interfering compounds (polyphenols, organic acids, pigments, proteases, etc.). Additionally, older leaves contain significant amounts of photosynthetic proteins (e.g.
Rubisco) which tend to interfere with the visualization of lower abundant proteins and dominate 2-DE gels.
Olive leaves are considered as one of the most difficult plant tissues to process. They contain high levels of phenolic secoiridoids (oleuropein) which turn into a very strong protein denaturant when the leaf is destroyed. This explains why direct homogenization of olive leaf in extraction buffers followed by protein precipitations results in brownish pellets which are recalcitrant to common protein extraction methods.
To address this issue, a protocol can be used to remove secondary metabolites from the leaf tissue prior to extraction. The said protocol combines TCA/acetone precipitation and phenol extraction to obtain well-resolved 2-DE patterns within 1 to 2 days. Powdered leaf tissue can also be homogenized with a polytron (Brinkman model PT3100) repeatedly at a maximum speed in cold 10% TCA/acetone plus 0.07% ME to produce good 2-DE maps. For less difficult tissues such as wheat leaves and rice leaf sheath and internodes, TCA/acetone precipitation can be used to achieve good 2-DE separation of proteins.
Fruits
Extracting proteins from fruits can present a unique challenge. Besides the fact that fruit tissues have low protein content (i.e. grapes and apples), they also contain significant amounts of interfering compounds such as phenolics, organic acids, lipids, pigments, and polysaccharides. Thankfully, protocols have been developed in recent years to successfully extract proteins from grapes, apples, bananas, strawberries, pears and olives.
Seeds
While seeds contain higher levels of proteins compared to other plant tissues, the significant amount of storage proteins, lipids (triacylglycerols) and carbohydrates (starch) in seed tissues often interfere with the extraction process.
In the case of castor seeds where lipids make up 50% of the seed dry mass, the protocol detailed here can be used to remove excess lipids from oil seed extracts prior to 2-DE. Protein extract of dry castor seeds was mixed with an equal volume of chloroform/methanol (2:1). Since chloroform weakens the forces between subunits of lipid–protein complexes, successful separation of proteins from lipids during subsequent TCA wash and precipitation steps can be achieved. The protocol also works well in other oil crop seeds such as olive, sunflower, rapeseed, and sesame.
When extracting total protein from peanut seeds, dried kernels are frozen in liquid nitrogen, ground and defatted with hexane at -20oC overnight before subjecting the sample in a modified TCA/acetone precipitation protocol.
Roots
The presence of secondary metabolites and vacuoles in root tissues presents a unique challenge when extracting total protein. However, it was observed that the TCA/acetone precipitation usually leads to a clean protein preparation and provides a protein yield equivalent to 0.1% of the fresh weight of the sample. Additionally, direct phenol extraction also produced high quality protein samples when used for maize and hemp roots.
Potato Tubers
It is interesting to note that while potato is considered as the 4th most important food crop in the world, extracting total potato tuber proteins via 2-DE still comes as a challenge for most researchers. In an effort to optimize the process, Delaplace et al. evaluated two protocols – one using protocols using SDS buffer/TCA/acetone precipitation and the other using phenol extraction. While the two methods produced similar extraction yields and total number of spots, the quality of resulting 2-DE gels of SDS extracts seem better. However, phenol extraction produced more proteins with Mr A > 40 kDa and more horizontal streaking on gels while SDS extraction produced more proteins with Mr A < 40 kDa.
