Mass spectrometry based proteomics

Gas chromatography mass spectrometry (GC-MS) was employed to determine the chemical composition of essential oil obtained from Eucalyptus globulus and Trachyspermum ammi. The essential oil obtained from leaves of Eucalyptus globulus and from seeds of Trachyspermum ammi by Clevenger apparatus. Chemical composition of the essential oils of E. globulus and T. ammi was analyzed by Gas chromatography – Mass spectrometry. Four main compounds in E. globulus were identified as Eucalyptol, Methyl –salicylate, Thymol, Para- cymene. 1, 8-cineole was found to be the main compound of E. globulus essential oil. Thymol, gamma- terpinene, para- cymene, beta- pinene were the main compounds of T. ammi oil and traces amount of beta myrcene, alpha- thujne, alpha- pinene, alpha- terpinene, carvacrol compounds of T. ammi were identified by GC-MS. Thymol was the main compound of T. ammi oil. The antifungal activity of E. globulus and T. ammi oils were screened against Trichophyton mentagrophytes and Epidermatophyton floccosum by using disc diffusion and modified micro dilution method. T. ammi oil shows highest inhibition zone in comparison to E. globulus oils and references antibiotics i.e. Clotrimazole and Ketoconazole.

Leaf proteome of Clematis chinensis, a traditional Chinese medicine (TCM) was analyzed by two-dimensional electrophoresis (2-DE) technique. The samples were extracted by phenol-SDS method (PSM) with high protein quantity i.e. 2.35±0.345 mg/g (yield/dw). Proteins were visualized by staining of gels by silver stain and CBB. The gel images of each species were compared by Image Master 2D Platinum software for analytical purpose. The 2-DE profile depicted distribution of 1085 spots and out of these only 255 protein spots (23.5%) were common to all analyzed taxa. The visualized protein spots showed pI range from 3.0 to 10.0 (pH) and M r of 7 kDa to 70 kDa. Twelve proteins were exclusively specific to C. chinensis when compared with its allies, C. finetiana and C. armandii, which may be used as biomarkers. Thirteen proteins were up-regulated in C. finetiana (0.75-0.95 fold) and twelve proteins in C. armandii (1.05-1.66 fold) whilst seven proteins down-regulated (0.66-0.94 fold) in former and three proteins (1.07-1.20 fold) in later one in comparison with C. chinensis. Twenty five differential and similar protein spots were picked and analyzed by LC-MS/MS technique. Identified proteins are related to energy metabolism (ATP synthesis), photosynthesis, environmental stimuli, regulating RNA metabolism, growth hormone regulators, evolutionary trends and gene expression. The efficiency and applicability of proteomic approach as biomarker for identification of C. chinensis is discussed in its quality control (QC) perspectives. Leaf proteins of Clematis plants are explored for the first time by 2-DE technique and debated for their metabolic role.

Escherichia coli RNA polymerase is a multi-subunit enzyme containing α(2)ββ'ωσ, which transcribes DNA template to intermediate RNA product in a sequence specific manner. Although most of the subunits are essential for its function, the smallest subunit ω (average molecular mass ∼ 10,105 Da) can be deleted without affecting bacterial growth. Creating a mutant of the ω subunit can aid in improving the understanding of its role. Sequencing of rpoZ gene that codes for ω subunit from a mutant variant suggested a substitution mutation at position 60 of the protein: asparagine (N) → aspartic acid (D). This mutation was verified at the protein level by following a typical mass spectrometry (MS) based bottom-up proteomic approach. Characterization of in-gel trypsin digested samples by reverse phase liquid chromatography (LC) coupled to electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) enabled in ascertaining this mutation. Electron transfer dissociation (ETD) of triply charged [(M + 3H)(3+)] tryptic peptides (residues [53-67]), EIEEGLINNQILDVR from wild-type and EIEEGLIDNQILDVR from mutant, facilitated in unambiguously determining the site of mutation at residue 60.