Protein Extraction from Leaves for Mass spectrometry

The Basics

Sample impurities can turn what should be easy-to-interpret mass spectra into a mess of peaks, but this can all be avoided with the optimal protein extraction method.

Plants are particularly challenging samples due to their rigid cell wall and abundance of non-protein components such as polysaccharides, lipids and phenolic compounds that can be detrimental to your downstream applications. The protocol you choose needs to remove most, if not all, of these non-protein components while maximizing protein retention.

Protein precipitation is a common way to obtain concentrated samples of pure protein for mass spec. Here are the general steps:

1. Grind the plant sample into a fine powder
2. Reconstitute that powder in a buffer
3. Force protein aggregation
4. Pellet the aggregates by using centrifugal force
5. Wash and dry the pellets
6. Re-solubilize the protein in your choice of buffer

The Preparation: Grinding the Leaves

After harvesting your sample, freeze it in liquid nitrogen to make it brittle and easy to grind into a fine powder. This is best accomplished via bead beating, but a budget-friendly alternative is using mortar and pestle.

When selecting your leaf preparation approach, you should consider how many samples you have, if you will be performing this method long term, your budget and level of urgency.

Bead beating is reliable, efficient, and often adaptable to automation or high-throughput methods, making it an ideal approach for thoroughly grinding your leaf samples. The trick is choosing the appropriate lysing matrix, which can vary in bead material, size, and shape. Certain beads are too aggressive for leaf materials and may shear proteins, whereas others are too weak.

MP Bio offers a variety of lysing matrices, with Lysing Matrix A and Lysing Matrix D suitable for protein extraction from plant leaves.

Pro Tips:

• Use fresh leaves and select leaves from the same location/region on the plant to minimize sample to sample variability.
• Store your homogenized sample(s) at −80 °C or use it immediately for protein extraction.
• Learn more about How to choose the right lysing matrix for your sample and the best lysing matrices for different types of agricultural samples.

The Methods: Protein Precipitation

Below are two top-notch protein extraction methods that are commonly used in plant proteomics and mass spectrometry:

TCA/acetone extraction: An ideal method if you do not need active protein.

• This method generally produces higher protein yield compared to phenol-based methods, but the range of proteins is lower. Extracted proteins could remain functional, but they are vulnerable, so it is best to use a different method if you need the structural integrity and protein activity to remain intact.
• Standard recipes¹:
  • Extraction solution: 10% TCA in acetone (ice cold) with 5 mM DTT
  • Wash solution 1: 10% TCA in acetone (ice cold)
  • Wash solution 2: 80% acetone (ice cold)

Phenol extraction: An ideal method for extracting a wider range of proteins.

• Compared with TCA/acetone, phenol extraction is more complex. It involves first performing a crude extraction using a well-buffered solution (e.g., 50 mM Tris-HCl, 5 mM DTT, and 1 mM PMSF), then a phenol extraction, and lastly a wash step (80% acetone)¹.
• This method better extracts more acidic and higher molecular mass proteins, but the total protein yield is often lower than that with the TCA/acetone method.

Both methods are highly effective at extracting major high-abundance proteins, but a particular method may better extract certain less-abundant proteins based on protein hydrophilicity. For instance, Wang et al. found that the phenol extraction method was most capable of extracting 3-β-hydroxysteroid dehydrogenase/isomerase (positive GRAVY value) from maize leaf midribs.

Pro Tips:

• Protect your proteins by using ice-cold reagents to slow protease activity throughout the extraction process.
• After your final wash step, it is imperative to dry the sample without drying it out completely. Overdrying will make solubilizing the sample very difficult or impossible, deeming your newly extracted proteins unusable.
• To dry your sample, discard the final wash solution—being careful not to disrupt the protein pellet—and let the sample air dry for 30 minutes. You’ll know your protein is sufficiently dry when you see a slight change in opacity.

References:

Wang, Ning et al. “Evaluation of Three Protein-Extraction Methods for Proteome Analysis of Maize Leaf Midrib, a Compound Tissue Rich in Sclerenchyma Cells.” Frontiers in plant science vol. 7 856. 14 Jun. 2016, doi:10.3389/fpls.2016.00856