Mass Spectrometric Investigations and Applications of Noncovalent Biomolecular Interactions
Date Issued
2006
Date
2006
Author(s)
Liau, Shing-Chih
DOI
en-US
Abstract
The thesis consists of two parts. In part I, we study dissociation of noncovalently bound heme from gaseous myoglobin ions by IRMPD with FT-ICR mass spectrometetry. In part II, we noncovalently coat polylysine on diamond nanocrystals for MALDI-TOF mass analysis of DNA oligoncleotides. The abstracts of both parts are shown below.
PART I: We have studied detachment of heme prosthetic groups from gaseous myoglobin ions by infrared multiphoton dissociation (IRMPD) by means of Fourier- transform ion cyclotron resonance (FTICR) mass spectrometry. Multiply charged holomyoglobin ions (hMbn+) were generated by electrospray ionization (ESI) and transferred to an ICR cell, where the ions of interest were isolated and fragmented by irradiation with 3-μm photons, producing apomyoglobin ions (aMbn+). Both of oxidized and reduced forms of myoglobin (Fe (III)-hMbn+ and Fe (II)-hMbn+) were invistegated
First, charged heme loss (with [Fe (III)-heme]+ and aMb(n-1)+ as the products) was detected for hMbn+ produced from a pure ferric myoglobin solution with different laser irradiation times. Next, both charged heme loss (with [Fe (III)-heme]+ and aMb(n-1)+ as the products) and neutral heme loss (with [Fe (II)-heme] and aMbn+ as the products) were detected concurrently for hMbn+ produced from a myoglobin solution pretreated with reducing reagents. The activation energies were determined by fitting these sets of experimental data separately to . With reference to Ea = 0.9 eV of charged heme loss for ferric hMbn+ determined by blackbody infrared radiation, a dissociation activation energy of 1.1 eV was deduced for the neutral heme loss of ferrous hMbn+ with n = 9 and 10.The difference in dissociation activation energy between these two channels is small, only ~25% (or 6 kcal*mol-1).
PARTII: In this experiment, the amine-terminated diamond nano- crystals (~100 nm in diameter) were prepared by noncovalent coating of carboxylated/ ox- idized diamonds with poly-L-lysines (PL), which form stable complexes with DNA oligonucleotides in solution. While the complexes are sufficiently stable to sustain repeated washing with deionized water, the DNA molecules can be readily eluted after incubation of the diamond adducts in aqueous ammonium hydroxide at elevated temperatures. No preseparation of PL or diamond nanocrystals is required for subsequent MALDI-TOF mass analysis.
A protocol based on amine-terminated diamond nanocrystals could be developed to isolate, concentrate, purify and digest DNA oligonucleotides in one pot for matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). It is shown that use of diamond nanocrystals as a solid-phase extraction support not only permits concentration of oligonucleotides in dilute solutions but also facilitates isolation of oligonucleotides from proteins in mixed solutions. Additionally, enzymatic digestions can be conducted on particle and, furthermore, the digests can be easily recovered from the solution to obtain base sequence information.
Subjects
質譜
肌血紅蛋白
奈米鑽石
Mass Spectrometry
FT-ICR
MALDI TOF
nanodiamond
myoglobin
Type
thesis
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