Chromatography Electrospray Ionization Quadrupole Research Articles

Plantlet formation via somatic embryogenesis and LC ESI Q-TOF MS determination of secondary metabolites in Butea monosperma (Lam.) Kuntze

Medicinal properties of Butea monosperma (BM) and overexploitation of bark as a rich source of flavonoids for different biological activities, development of efficient method for high frequency somatic embryos and in vitro synthesis of bioactive secondary metabolites using plant tissue culture technology is important. Initially, callus was induced from leaf explants of BM on Murashige and Skoog (MS) medium containing 0.25 mg L−1 2,4-d-dichlorophenoxyacetic acid (2,4-d) with 0.1 mg L−1 kinetin (Kn) and ascorbic acid (AA). MS half strength macronutrients and full strength micronutrients containing 0.25 mg L−1 2,4-d with 0.1 mg L−1 Kn, and 0.5 mg L−1 AA provided fragile callus with 84.0 ± 1.00 % optimal growth response. Shoot formation occurred via somatic embryogenesis through an intermediary callus phase. However, 2.1 mg L−1 thidiazuron with 0.5 mg L−1 AA provides high frequency (79.6 ± 2.02 %) of somatic embryogenesis within 5 weeks. Developed embryos when transferred to woody plant medium containing 0.5 mg L−1 AA with 3.0 mg L−1 Kn and 0.5 mg L−1 α naphthalene acetic acid responded 44.0 ± 0.00 % embryo maturation, whereas 0.5 mg L−1 Kn, 0.3 mg L−1 indole-3-butyric acid, and 0.25 mg L−1 AA induced rooting within 6 and 8 weeks, respectively. Liquid chromatography electro spray ionization quadrupole time of flight mass spectrometry (LC ESI Q-TOF MS) analysis of in vitro cultures showed similarity to those compounds identified in wild grown leaf samples known for osteogenic activity. Histological investigation through scanning electron microscopy demonstrates the developmental stages of somatic embryos, shoot bud formation, and induction of root primordial.

Protein profiling in hepatocellular carcinoma by label-free quantitative proteomics in two west African populations.

BackgroundHepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.MethodsMass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.ResultsTwenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.ConclusionsThe validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance.

Abstract 3301: Tumor biomarkers in mammary adenocarcinoma cell clones of different metastatic potential

Abstract Changes in the pattern of protein expression by tumor cells reflect the stage of tumor progression, the potential for invasion and metastasis, and drug resistance. Objectives: To use proteomic analysis to detect differences in protein expression that serve as biomarkers of tumor cell properties and to translate the results into diagnostic and prognostic tools based on immuno-cytological staining and immunoassay. Methods: Two-dimensional isoelectric focusing and gel electrophoresis (2-DG) followed by mass spectrometry were used to detect, measure, and identify changes in protein expression. MTC is a non-metastatic cell clone derived from a primary rat mammary adenocarcinoma. MTLn2 and MTLn3 are low and high metastatic potential cell clones derived from lung metastases of the primary tumor. 2-DG was performed simultaneously on samples of 3 extracts from cultured cells of each of the 3 clones. The 9 gels were stained with SYPRO Ruby. Matching of protein spots, densitometric analysis, and statistical analysis were performed using PDQuest 7.1 software. Excised proteins were identified by comparing the mass and sequence of tryptic peptides with those of all known rat proteins using liquid chromatography electrospray-ionization quadrupole ion trap mass spectrometry, SEQUEST software, and the NCBI protein database. Results: A total of 1500 proteins were detected. The patterns of protein expression of MTLn2 and MTLn3 cells were similar. Only 5 spots had a greater than 3-fold difference in staining intensity that was statistically significant (p<0.05), whereas 48 spots differed between MTC and MTLn3 cells. Twenty spots were selected for further study, 10 that had a positive correlation of staining intensity with metastatic potential and 10 that had a negative correlation. Of the 17 unique proteins that were identified, five were of special interest because they are often cited as potential tumor biomarkers. These included the positive biomarkers nucleophosmin (NPM) and protein 14-3-3 sigma and the negative biomarkers raf kinase inhibitor protein (RKIP), peroxiredoxin-2, and galectin-1. Immuno-cytological staining and ELISA confirmed increased NPM and protein 14-3-3 sigma expression and decreased galectin-1 expression. The results suggest a role for stromal elements in biomarker expression. Conclusions: Increased metastatic potential is associated with positive and negative changes in protein expression. Proteins that are positively correlated with metastatic potential may be more useful as clinical biomarkers, whereas those with negative correlations may provide information about underlying mechanisms of metastatic spread and possible stromal influences. Changes in protein expression with differences in tumor properties will provide a consensus panel of biomarkers for diagnostic and prognostic tests. Supported in part by the Sam Mount, Jr. Endowment for Oral Cancer Research. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3301.