Proteomics & Genomics / General Biochemistry

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    (2013-04-12) Sahyouni, Fatima
    Purpose: To identify estrogen-induced differential protein expression in rat uterine tissue using a rapid label-free proteomics and systems biology approach. Methods: Ovariectomized rats were treated short-term with subcutaneous E2 injections using corn oil as a vehicle. Approximately 10 mg of tissue were dissected from the uterus of vehicle-treated control and E2-treated animals for proteomic analyses. Uterine proteins were extracted with 8M urea for 30 minutes and subsequently processed by reduction, alkylation and digestion for mass spectrometry analysis. The samples were analyzed using a hybrid linear ion trap-Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source and connected to a nanoflow liquid chromatography system. MS/MS data was searched against a composite IPI rat protein database containing both forward and randomized sequences using the Mascot software. Quantitation was performed using an MS/MS-based total ion currents (TICs) approach using the Scaffold software. Additionally, Ingenuity Pathway Analysis (IPA) was utilized to derive interaction networks among the identified proteins. Results: The mammalian uterus increases its weight due to fluid imbibition and cell proliferation by exogenously administered estrogenic compounds. With the observation of weight gain in the treated uterus compared to non-treated control rats, we confirmed E2's uterotrophic effects for our subsequent proteomics study. Out of a total of 262 identified proteins, 163 proteins were differentially regulated (with p<0.05 considered statistically significant) by the hormone. Of the 163 proteins that were significantly regulated, 153 were up-regulated in E2-treated uteri and 10 were down-regulated in E2-treated uteri. These 163 proteins were submitted and mapped into 19 networks that merged into E2-regulated pathways. Top networks included molecular transport, carbohydrate metabolism, cancer, developmental disorders and cellular function and maintenance. Implicated diseases were endocrine system and metabolic disorders. Top signaling pathways involved metabolic pathways, steroid signaling and actin cytoskeleton signaling. Conclusions: In addition to the expected increase in wet uterine weights, we have elucidated and organized a large number of E2-induced protein expression changes into interaction networks. Metabolism and developmental disorders were implicated as the top networks. (Supported by the Robert A. Welch Foundation, BK-0031, and the NIH grant AG031535)
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    (2013-04-12) Talamantes, Tatjana
    Purpose: Identification of carotenoid-regulated proteins in cyanobacteria by mass spectrometry-based proteomics. Methods: Strains were derived from Synechocystis sp. PCC 6803 (wild type, WT). WT and ΔcrtH/B mutant cells were cultivated at 30℃ in light-activated heterotrophic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on crude cellular protein extracts, and gels were cut horizontally into pieces for subsequent in-gel tryptic digestion and reversed-phase liquid chromatography (LC) coupled to data-dependent electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) using nanoLC (Eksigent) and LTQFT (Thermo) instruments. MS/MS spectra were extracted by BioWorks and searched against a composite protein sequence database of cyanobacteria using Mascot. Quantitative protein expressions were obtained through MS/MS spectral counts determined through Scaffold and by extracted peptide intensity features generated with the Progenesis LC-MS software. Results: Using the Synechocystis sp. PCC 6803 ΔcrtH/B mutant, our "GeLC-MS/MS" analyses permitted global-scale identification of protein expression changes in carotenoid-regulated cells by label-free approaches. Overall, 497 proteins were identified in this genetically modified strain of cyanobacteria. Considering at least a 2-fold change using the total number of MS/MS spectra identified for a particular protein (Scaffold), 60 carotenoid-regulated proteins were obtained from G-tests (p<0.05). As an alternative data-processing method, Progenesis LC-MS was used to process the same set of raw data files by alignment and normalization of MS peak intensities, data filtering for charge states and, then, exporting peak lists to query against the cyanobacteria protein database using Mascot. When requiring at least a 2-fold change in expression and excluding hypothetical hits, this latter approach afforded 197 carotenoid-regulated proteins by analysis of variance (ANOVA, p<0.05). From genome information on Synechocystis (a part of CyanoBase, an online resource for cyanobacterial genomes), these proteins were found to be involved in photosynthesis and respiration, transport and binding, transcription and translation, and biosynthesis. Conclusions: Altogether, our GeLC-MS/MS analyses permitted a successful quantitative survey of carotenoids' global impact on differential expressions of key cellular proteins in the ΔcrtH/B mutant versus WT Synechocystis sp. PCC 6803 for the first time.
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    (2013-04-12) Zhang, Fan
    Purpose: Alternative splicing is an important widespread mechanism for generating protein diversity and regulating protein expression. In human cells, about 40-60% of the genes are known to exhibit alternative splicing. Recent methodological advances, including EST sequencing, exon array, exon-exon junction array, and next-generation sequencing of all mRNA transcripts, have made it possible to perform high-throughput alternative splicing analysis. However, high-throughput identification and analysis of alternative splicing in the protein level has several advantages. For example, mRNA abundance in a cell often correlates poorly with the amount of protein synthesized, and proteins rather than mRNA transcripts are the major effector molecules in the cell. The combination of alternative splicing database and tandem mass spectrometry provides a powerful technique for identification, analysis and characterization of potential novel alternative splicing protein isoforms from proteomics. Therefore, we used a three steps pipeline to create an synthetic alternative splicing database(SASD) for tandem mass spectrometry data analysis. Methods: First we derived exons and introns from UCSC Genome Database, then we analyzed six types of combinations of exons and introns for the transcription of artificial splicing gene (exon_exon_normal, exon_exon_skipping, intron_exon, exon_intron, single exon, and single intron), and lastly we performed the translation of the artificial transcripts. Results: In addition, we built a web interface for users to browse 1) by genes/proteins, 2) by biological process, 3) by signaling and metabolic pathway, 4) by disease, 5) by drug, and 6) organ. Lastly, we presented two case studies: 1)in breast cancer and 2) in liver cancer, to demonstrate that the SASD can enable users to analyze, characterize, and understand the impact of alternative splicing on genes involved in drug, disease, pathway, function, and organ-specificity. Conclusions: The SASD provides the scientific community with an efficient means to identify and characterize novel Exon Skipping, Intron Retention, and alternative 3' splice site and 5' splice site protein isoforms from mass spectrometry data. We believe that it will be useful in annotating genome structures using rapidly accumulating proteomics data and assist scientific research on signal transduction pathways regulating pre-mRNA, clinical therapy, disease prevention, and drug development.