Proteome profiling of Cumin by Q-Exactive HF mass spectrometer
A shotgun proteomic approach was used to obtain comprehensive dataset of cumin proteins. For this purpose we applied a one dimensional electrophoresis coupled with high resolution mass spectrometry. The first step in our proteome analysis strategy, we evaluate different extraction methods, based on differential solubility of protein. Among those methods, Readyprep protein extraction kit provide high purity of protein extract, gave higher protein yield and typically good resolution of protein bands on one dimensional gel electrophoresis. Further nanoLC-MS/MS analysis by state-of-the-art Q Exactive HF mass spectrometer resulted in identification of very high number proteins. Bioinformatics analysis provide functional distribution of identified protein. To our knowledge, this is the first study aimed for in depth study of cumin proteome.
Sample Processing Protocol
In present study we compared three different extraction methods for protein sample preparation. First method utilize sodium acetate extraction at slightly acidic conditions (pH 5.5) and ammonium sulfate (SA/AS) precipitation. Second method uses Tris saturated Phenol solution for protein extraction followed by precipitation with methanolic ammonium acetate. We also used Readyprep total protein extraction kit from BioRad and evaluate its suitability for cumin proteins. Total extracted proteins was separated on one-dimensional gel electrophoresis (1-DE) and the entire gel lane was excised and divided into slices prior to the proteolytic digestion using trypsin. Peptides collected from tryptic digestion different slices, were subjected to further analysis on nanoLC system prior to MS/MS analysis. The use of modern state-of-the-art Q Exactive HF mass spectrometer allowed identification of several proteins.
Data Processing Protocol
All raw data files from Xcalibur software were directly analyzed by MaxQuant (version 18.104.22.168) using the Andromeda Search engine against the Viridiplantae database from UniProt. The intensity-based absolute quantification (iBAQ) in MaxQuant was performed on the identified peptides to quantify protein abundance.
Zaman U, Urlaub H, Abbasi A. Protein Profiling of Non-model Plant Cuminum cyminum by Gel-Based Proteomic Approach. Phytochem Anal. 2017 PubMed: 29148164