Browsing by Subject "alternative splicing"
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Item Identification of novel alternative splicing biomarkers for breast cancer with LC/MS/MS and RNA-Seq(BioMed Central Ltd., 2020-12-03) Zhang, Fan; Deng, Chris K.; Wang, Mu; Deng, Bin; Barber, Robert C.; Huang, GangBackground: Alternative splicing isoforms have been reported as a new and robust class of diagnostic biomarkers. Over 95% of human genes are estimated to be alternatively spliced as a powerful means of producing functionally diverse proteins from a single gene. The emergence of next-generation sequencing technologies, especially RNA-seq, provides novel insights into large-scale detection and analysis of alternative splicing at the transcriptional level. Advances in Proteomic Technologies such as liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), have shown tremendous power for the parallel characterization of large amount of proteins in biological samples. Although poor correspondence has been generally found from previous qualitative comparative analysis between proteomics and microarray data, significantly higher degrees of correlation have been observed at the level of exon. Combining protein and RNA data by searching LC-MS/MS data against a customized protein database from RNA-Seq may produce a subset of alternatively spliced protein isoform candidates that have higher confidence. Results: We developed a bioinformatics workflow to discover alternative splicing biomarkers from LC-MS/MS using RNA-Seq. First, we retrieved high confident, novel alternative splicing biomarkers from the breast cancer RNA-Seq database. Then, we translated these sequences into in silico Isoform Junction Peptides, and created a customized alternative splicing database for MS searching. Lastly, we ran the Open Mass spectrometry Search Algorithm against the customized alternative splicing database with breast cancer plasma proteome. Twenty six alternative splicing biomarker peptides with one single intron event and one exon skipping event were identified. Further interpretation of biological pathways with our Integrated Pathway Analysis Database showed that these 26 peptides are associated with Cancer, Signaling, Metabolism, Regulation, Immune System and Hemostasis pathways, which are consistent with the 256 alternative splicing biomarkers from the RNA-Seq. Conclusions: This paper presents a bioinformatics workflow for using RNA-seq data to discover novel alternative splicing biomarkers from the breast cancer proteome. As a complement to synthetic alternative splicing database technique for alternative splicing identification, this method combines the advantages of two platforms: mass spectrometry and next generation sequencing and can help identify potentially highly sample-specific alternative splicing isoform biomarkers at early-stage of cancer.Item SASD: THE SYNTHETIC ALTERNATIVE SPLICING DATABASE FOR IDENTIFYING NOVEL ISOFORM FROM PROTEOMICS(2013-04-12) Zhang, FanPurpose: 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.