Abstract
The unique properties of inorganic nanoparticles (NPs) and their interaction with pulsed laser irradiation have been exploited in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the enhanced desorption and ionization of carbohydrates or glycans. Glycoconjugates are ubiquitously present and play critical roles in various biological processes. Due to their low stability and incredibly high degree of structural diversity, the structural characterization of glycans generally requires chemical derivatization and sophisticated instrumentation. However, in most cases, the derivatization requires large sample amount and enhances the risk of introducing contaminants and side reactions. The development of rapid, sensitive and comprehensive glycan sequencing methods that do not require chemical derivatization remains a considerable challenge. In this thesis, the development of single-step pseudo-MS/MS approach for tunable ionization and fragmentation to facilitate structure determination of glycoconjugates, using concentration-dependent UV-absorbing matrix-functionalized magnetic nanoparticles and MALDI MS was presented. Without chemical derivatization, this approach successfully distinguished isomeric sets of trisaccharides. Low concentration of nanomatrix provided enhanced signal for accurate mass determination of intact molecular ions in the sample. In contrast, high concentration of nanomatrix induced extensive and unique fragmentation, including high-energy facile bond breakage (A- and X-type cross-ring cleavages), which facilitated the linkage and sequence characterization of oligosaccharides without conventional tandem mass spectrometric instrumentation. The practicality of this approach for complex sample analysis was evaluated by an oligosaccharide mixture, wherein molecular ions are unambiguously observed and signature product ions are distinguishable enough for molecular identification and isomer differentiation. Subsequently, the roles of the multilayer nanomatrix components: matrix (energy absorption), silane-coating (energy pooling and dissipation) and core Fe3O4 (fragmentation) was also investigated. The plausible electron and energy transfer mechanism was proposed based on the threshold energy of photoelectrons and thermal energy measurements. The differentiation of tri-oligosaccharides, which served as the first step toward glycan characterization by nanoparticle-assisted MALDI-MS, had shed some insight on the nanoparticle-mediated energy transfer dynamics behind the proposed approach. The structure-specific fragmentation of molecular ions in mass spectrometry provides an efficient analytical method for confirming unknown analytes or for elucidating chemical structures. Next, a method for complicated glycan characterization in a single assay by employing the 2,5-dihydroxybenzoic acid functionalized mercury telluride nanoparticles (HgTe@DHB NPs) as a dual ionization-dissociation element in MALDI-MS was developed. Using a linear glycan, HgTe@DHB NPs promote laser-induced extensive dissociation and intensive detection of the target glycan, superior to the HgTe microparticles and other functionalized and non-functionalized inorganic nanoparticles (TiO2, ZnO, and Mn2O3 NPs). Abundant generation of diagnostic glycosidic (Y-, and B-type ions) and cross-ring cleavage (A-type ion) ions permit unambiguous determination of the composition, sequence, branching, and linkage of labile sialylated glycans. The general utility of this approach was demonstrated on the characterization of labile sialylated glycans and two sets of complicated isomeric glycans. Our results show that this "pseudo-MS/MS” obtained by HgTe@DHB can be beneficial for the analysis of biologically relevant and more complicated glycans without the need of chemical pre-derivatization and conventional tandem mass spectrometry. Lastly, a facile and innovative technique for glycopeptide characterization incorporating the microwave-assisted enzymatic deglycosylation and nanoparticle-assisted LDI-MS was designed and demonstrated. The mixture of glycopeptide, glycan, and peptide resulted from microwave-assisted deglycosylation reaction was subsequently analyzed in MALDI-MS. Using MNP@DHB matrix, the composition, sequence, branching, and linkage information of fucosylated N-glycoconjugates were acquired, while, the sequence of the peptide backbone was obtained by tandem mass spectrometry technique. The applicability of this method was shown by its ability to distinguish two synthetic isomeric N-glycopeptides. Through the observed unique glycosidic bond (B3 ion, m/z = 536.8) and cross-ring cleavage (2,4A6, m/z = 1324.0) ions, the terminal fucosylated glycopeptide can be unambiguously discriminated from core fucosylated glycopeptide. It is expected that the results of this work can contribute to the ever expanding field of analytical chemistry, as well as advance the understanding in nanoparticle-based MALDI mass spectrometry for detection and characterization of glycans and glycoconjugates.