Author : Zhen Sun
Publisher :
ISBN 13 :
Total Pages : 78 pages
Book Rating : 4.:/5 (812 download)
Book Synopsis Studies of Atmospheric Pressure Visible-wavelength MALDI-MS by : Zhen Sun
Download or read book Studies of Atmospheric Pressure Visible-wavelength MALDI-MS written by Zhen Sun and published by . This book was released on 2012 with total page 78 pages. Available in PDF, EPUB and Kindle. Book excerpt: Matrix-assisted laser desorption/ionization technique (MALDI) is well proven as a soft and efficient ionization technique used in mass spectrometry (MS) for the analysis of biomolecules (such as sugars, peptides, DNAs, and proteins), and large organic molecules (such as synthetic polymers). While the technique has been developed in 1980s, the improvements in MALDI-MS instrumentation and search for new matrices have continued. In the first project of this thesis, an atmospheric pressure (AP) visible-wavelength MALDI source was developed and coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer. This instrument employed a pulsed laser emitting light at a wavelength of 532 nm to desorb and ionize samples such as dyes, oligosaccharides, peptides, and synthetic polymers at atmospheric pressure. Ions formed were analyzed by the Q-TOF in both MS and tandem MS (MS/MS) modes. Several visible-wavelength absorbing dyes were successfully utilized and studied as matrices. Among these dyes, nuclear fast red (NFR), rhodamine B isothiocyanate (RITC), and 5(6)-carboxytetramethyl-rhodamine N-succinimidyl ester (TAMRA, SE) are reported as effective visible-wavelength MALDI-MS matrices for the first time. This project demonstrates applicability of visible-wavelength AP MALDI-MS and AP MALDI-MS/MS to the detection and structural analysis of dye molecules, biomolecules such as sugars and peptides, and synthetic polymers. In the second project, the model protein lysozyme was modified with fluorescein isothiocyanate (FITC). 64% of lysozyme was conjugated with FITC. Crystallization of pure lysozyme and FITC-conjugated lysozyme were performed after protein purification and pre-concentration. Pure lysozyme samples formed very good crystals, but there was no crystal observed for FITC-conjugated lysozyme. These two samples were then analyzed by different sample preparation methods using MALDI-MS. However, only lysozyme crystal with UV MALDI matrix spotted on top of it worked well. By performing a series of experiments, we confirmed that: it is crucial to have a matrix for the analysis of a single lysozyme crystal and the lysozyme crystal has different crystal structure than the matrix-analyte co-crystal. Secondly, it has not been possible to form crystal with 64% of lysozyme being conjugated with FITC dye. In summary, we developed an AP visible-wavelength MALDI-MS methodology, which enabled the analysis of biomolecules and synthetic polymers. Visible-wavelength AP MALDI-MS and MS/MS analyses of a variety of samples at atmospheric pressure were presented for the first time. Several visible-wavelength absorbing dyes were successfully utilized as matrices, and three dyes were reported as novel visible-wavelength MALDI matrices. Additional experiments were performed to covalently attach FITC to lysozyme and crystallize fluorochrome-conjugated protein. MALDI-MS was employed to study crystals of unlabeled lysozyme, while crystallization of FITC-conjugated lysozyme needs to be optimized further.
