The Crop Proteome Engine
To create our Proteome Atlas of the world’s 100 most important crops, we have developed an efficient and streamlined protocol
- Tissue Disruption and Metabolite Extraction: Mechanically disrupt the tissue, followed by metabolite extraction and protein precipitation using a solution of 10% trichloroacetic acid/acetone, tris-buffered sodium dodecyl sulphate (SDS), and phenol.
- Protein quantification: using a BCA analysis.
- On-Bead Digestion and Desalting: Perform on-bead digestion using trypsin (SP3 method) and desalt the samples via hydrophobic solid-phase extraction.
- High pH Reversed-Phase Chromatography: Separate the digested peptides using high pH reversed-phase chromatography.
- LC-MS/MS Analysis: Conduct online micro-flow liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the fractions over a 60-minute gradient.
- Protein Identification and Quantification: Identify proteins using MaxQuant/Andromeda with Prosit rescoring, and perform quantification using MaxQuant.