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Detection and sequencing of microRNA using MALDI time-of-flight mass spectrometry

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Chue Hue Yang (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Norman Chiu

Abstract: The discovery of microRNAs (miRNAs) and their abilities to regulate in vivo protein synthesis have led to growing interests in miRNA research. Due to their functions on regulating cellular processes that are related to diseases such as cancer, miRNAs can potentially become a new class of diagnostic biomarkers and therapeutic agents. Since early 2000, researchers have reported more than 1,000 human miRNAs. To facilitate high-throughput clinical studies, there are needs for more accurate and robust analytical methods to detect and quantitate miRNAs. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used mainly for proteomics research. In the past two decades, MALDI-TOF MS has also been used in genomics research. Specifically, MALDI-TOF MS has been used in the analysis of oligonucleotides, both DNA and RNA. In this study, the use of MALDI-TOF MS to detect specific miRNA was optimized, and the applicability of tandem MALDI-TOF/TOF to perform partial de novo sequencing was evaluated. The MALDI process requires a small organic compound, often known as "matrix", which readily undergoes desorption on contact with UV laser and assists the ionization of analyte. Therefore, an investigative study was designed to compare five commonly used MALDI matrix compounds for oligonucleotide analysis. The selected matrix compounds includes 3-hydroxypicolinic acid (3-HPA), 2',3',4'-trihydroxy-acetophenone (THAP), 6-aza-thiothymidine (6-ATT), 3,4-diaminobenzophenone (DABP) and 3-hydroxycoumarin (3-HC). The goal is to identify which is the best matrix for supporting the MALDI process and the subsequent tandem MALDI-TOF/TOF measurements. The 4700 Applied Biosystems Proteomics Analyzer was initially used to perform linear MALDI-TOF MS analysis of a selected microRNA standard (miR-124a). The initial results of our MS/MS measurements indicated higher signal intensity or ion count of the parent ion of miRNA is required to achieve sequencing. For this reason, we embarked on a series of studies to increase the signal intensity, especially on the effects of various parameters that were related to the desorption of miRNA during the MALDI process. The results indicated 3-HPA matrix has provided the highest signal intensity. Thus, 3-HPA was used to further optimize the MALDI-TOF MS measurements of miRNA. Once the parameters of MALDI-TOF MS were optimized, the use of tandem MALDI-TOF/TOF MS to perform partial de novo sequencing of miRNA was evaluated. Following the acquisition of initial MS/MS spectra of miR-124a, the use of different collision-induced dissociation (CID) pressures and delayed times to induce the fragmentation of miR-124a parent ion were investigated. It was determined that the 4700 Proteomics Analyzer could have a limitation on measuring the singly-charged miR-124a parent ion (7,161 m/z). This could be due to the current design and/or settings on the reflectron within the MALDI-TOF/TOF instrumentation were not suitable for measuring ions with high molecular masses. To overcome this limitation, the doubly-charged miR-124a parent ion (3,582 m/z) was selected as an alternative for performing the tandem MALDI-TOF/TOF MS measurements. Both post-source decay (PSD) and CID in the positive ion mode were used.

Additional Information

Publication
Thesis
Language: English
Date: 2010
Keywords
3-hydroxy picolinic acid, Collision-induced dissociation, Fragmentation, MALDI-TOF MS, MicroRNA, Sequencing
Subjects
Small interfering RNA.
Mass spectrometry.