Session: Session 3

Overcoming the artefactual oxidation in SPEED extraction for leaf samples

A new universal method for the rapid extraction and digestion of proteins, called "Sample Preparation by Easy Extraction and Digestion" (SPEED), has recently been developed [1]. This method consists of three steps: acidification (addition of trifluoroacetic acid (TFA)), neutralisation (addition of tris(hydroxymethyl)aminomethane (TRIS)) and digestion. This protocol addresses several challenges in proteomics analysis by mass spectrometry, such as being a detergent-free method and allowing a very rapid sample processing. Its effectiveness has been demonstrated in animal and bacterial cells. However, this protocol has not yet been tested for plant cells. We have started to test this protocol on Arabidopsis thaliana leaves, root and seeds and showed that oxidation levels in the leaves are much higher than in conventional extraction methods (approximately 80% vs 35%, respectively). To overcome this challenge, we tested different scavenging and antioxidant molecules at different stages of the protocol to determine when oxidation occurs and which is the best candidate to reduce artefactual oxidation. Preliminary results showed that methionine applied during acidification and neutralisation reduce the levels of oxidation, but further testing was required



[1] Doellinger et al. (2020) Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid Extraction. Mol Cell Proteom.(1):209-222. doi: 10.1074/mcp.TIR119.001616.