Differential proteomics of two varieties of Casearia sylvestris Sw. (Salicaceae)

The Brazilian native plant species Casearia sylvestris SW. (Salicaceae) is an important representative of the Casearia Jacq. genus due to its antiulcerogenic, anti-inflammatory and cytotoxic activities. This species is divided into two varieties according to its morphology. Interestingly, C. sylvestris variety Sylvestris can be characterized by the production of clerodane-type diterpenes (casearins), while C. sylvestris variety Lingua produces mainly phenolic compounds [1]. Despite the existence of data concerning its pharmacological properties, secondary metabolite chemistry and morphological features, so far nothing is known about its expressed proteins. Thus, the aim of this work was to access the protein profile of C. sylvestris by selecting individuals that show differentiated production of diterpenes and phenolic compounds. To achieve this goal, five trees of each variety were sampled to compose a representative sampling and their metabolic profiles were analysed by ultra-performance liquid chromatography coupled to diode-array-detection (UPLC-DAD). Main peaks identification was achieved by liquid chromatography coupled to mass spectrometry (LC-MS) or by comparison using standard compounds. Protein extracts from both varieties were fractionated using gel-free digestion and fractionation (FASP) [2] followed by liquid chromatography coupled to tandem mass spectrometry analyses (LC-MS/MS). Protein identification and comparison between the two varieties were made in the peptide level using the platform Proteomatic® [3], running OMSSA and X! Tandem against the Populus trichocarpa protein data bank, and PEAKS for de novo amino acid sequencing from MS/MS data. Using this approach, it was possible to identify 820 peptide sequences and to annotate 547 proteins with high accuracy. In conclusion, a combination of metabolic profiling with large scale proteomic study of C. sylvestris allowed the detection of considerable differences between the two varieties.

Keywords: Proteomics, Metabolomics, Casearia sylvestris Sw, LC-MS/MS

References:

[1] Claudino JC, Sacramento LVS, Koch I, Santos HA, Cavalheiro AJ, Tininis A, Santos AG. Evaluation of morpho-anatomical and chemical differences between varieties of the medicinal plant Casearia sylvestris Swartz.

An Acad Bras Cienc 2013; 85(4): 1253 – 1265.

[2] Wisniewski JR, Zougman A, Mann M. Combination of FASP and stagetip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. Nat Methods 2009; 6:359 – 362.

[3] Specht M, Kuhlgert S, Fufezan C, Hippler M. Proteomics to go: Proteomatic enables the user-friendly creation of versatile MS/MS data evaluation workflows. Bioinformatics 2011; 27 (8): 1183 – 1184.