Two-dimensional mapping as a tool for classification of green coffee bean species

• Published in: Proteomics (Weinheim) 2005-02, Vol.5 (3), p.710-718
• Main Authors: Gil-Agusti, Maria Teresa, Campostrini, Natascia, Zolla, Lello, Ciambella, Corrado, Invernizzi, Carlo, Righetti, Pier Giorgio
• Format: Article
• Language: English
• Subjects: Chromatography, High Pressure Liquid; Coffea - chemistry; Coffea - classification; Coffea arabica; Electrophoresis, Gel, Two-Dimensional; Green coffee beans; Peptide Mapping; Plant Extracts - chemistry; Plant Proteins - analysis; Proteome - analysis; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Two-dimensional maps
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200401014

Two species of the genus Coffea, Coffea arabica (Colombia) and Coffea canephora (Indiano Robusta) were analysed by two‐dimensional (2‐D) maps in order to obtain fingerprints of the expressed polypeptide chains and to determine which ones would characterize the two species. Green beans were milled under liquid nitrogen. A dry powder was produced by three different extraction protocols aimed at eliminating interfering substances (polyphenols). A reduced powder was produced by two successive extractions performed in acetone. Trichloroacetic acid (TCA; 10% w/v) and β‐mercaptoethanol (0.07% v/v) in acetone were used for the first extraction (a) and 10% w/v TCA in acetone was used for the second extraction (b). Proteins were then solubilized in a solution (40 µL per 1 mg powder) containing 7 M urea, 2 M thiourea, 3% w/v 3‐(3‐cholamidopropyldimethyl‐amino)‐1‐propanesulfate, 1% v/v carrier ampholytes, 40 mM Tris, 5 mM tributylphosphine and 10 mM acrylamide as alkylating agent. Following incubation at room temperature for 1 hour and centrifugation (7000 rpm for 20 minutes), the supernatant was used for 2‐D electrophoresis. The proteins were revealed by Sypro Ruby staining. Master maps of the five replicas of each species were compared by PDQuest analysis. The results of this differential proteome analysis were: sixteen proteins were expressed solely in C. canephora (var. Indiano Robusta) and five proteins were only found in C. arabica (var. Colombia). Another eight proteins were up‐regulated in C. canephora (var. Indiano Robusta) in comparison to C. arabica (var. Colombia) and one was down‐regulated in the same comparison. A number of these polypeptide chains were further characterized by mass spectrometry in the matrix‐assisted laser desorption/ionisation‐time of flight mode. Additionally, considering the low number of protein sequences of Coffea present in the databases we also investigated some spots with a more powerful tool, reversed phase‐high‐performance liquid chromatography‐electrospray ionisation‐tandem mass spectrometry, thus obtaining an internal peptide sequence. The general properties of the identified proteins are presented and discussed.