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Derivatization of Amino Acids for Surface Assisted Laser Desorption/Ionization (SALDI) Mass Spectrometry Analysis

  • Author / Creator
    Ji, Jing
  • Metabolites with low molar mass, such as amino acids, are of great importance in disease diagnosis. The rapid and accurate detection of amino acids and other small metabolites in biological samples has become significantly important. Surface assisted laser desorption/ionization (SALDI) has been applied in this area for detecting small metabolites, and nanostructure-based surfaces for SALDI provide reduced background noise and improved intensity in mass spectrometry. However, salts and other matrices in biological samples are a key challenge for accurate quantification of amino acids. In this thesis, we describe an approach to matrix-free SALDI strategies on nanoporous silicon surfaces for quantitative analysis of amino acids in complex biofluid samples. The approach removes interference through a simple on-chip cleanup method utilizing hydrophobic interactions. This thesis investigates the application of nanoporous silicon films fabricated by glancing angle deposition (GLAD) technique for matrix-free SALDI mass spectrometry. The GLAD chips have controllable morphology, stable chemical property, and long shelf life, which are suitable for SALDI-MS. We investigate a silanization method to perfluoro modify the surface of GLAD chips, and utilize the affinity of perfluoro coated surfaces to fluorous labeled analytes for on-chip purification. In chapter 2, different fluorous propanol as derivatizing reagents are tested to label carboxyl groups in amino acids, followed by SALDI-MS measurements on perfluoro coated GLAD films. The fluorous derivatization raises the mass of analytes to less background-obscured higher mass regions, and enables separation of fluorous labeled compounds from unlabeled ones on perfluoroalkyl-silylated GLAD chips through F-F interactions. A simple on-chip cleanup step is applied to remove unlabeled compounds, while the fluorous derivatives are retained, resulting in improved sensitivity in SALDI-MS. In chapter 3, the fluorous propanol based derivatization reaction is used for the quantification of amino acids in serum. C18 beads solubilized in methanol are used to remove proteins from the serum samples, the samples are then labeled by 3-(perfluorohexyl)propan-1-ol and measured by SALDI-MS. The quantitative results show good consistency with normal human ranges, and compare well with other studies. In the second segment of this thesis, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) and 1H-perfluorohexane-1,1-diol are evaluated as the derivatization reagents to label amine groups in amino acids. The quantitative results of 1H-perfluorohexane-1,1-diol labeled derivatives provide reliable linear regression, and the relative standard deviations in the intensity ratios between the analyte and the internal standard are low, indicating a great potential for precise quantitative analysis of amino acids in real samples.

  • Subjects / Keywords
  • Graduation date
    Fall 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
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  • License
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