Method Development Of Chemical Derivatization For N-Linked Glycan Analysis By Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Glycans have unique characteristics that are significantly different from nucleic acids andproteins in terms of biosynthesis, structures, and functions. N-glycosylation of proteinsplays multiple key roles in biological systems, including cell adhesion, signal transductionand molecular recognition. Differential N-linked-glycan profiling may provide overview ofvariations between healthy and diseased individuals for identifying novel prognostic anddiagnostic markers of disease, such as neurodegenerative diseases, metabolic disease andcancer. Many techniques have been used for this purpose, in which, mass spectrometry (MS)has been regarded as the most powerful one for its high sensitivity and accuracy inidentification and structure analysis of glycans. However, their isomeric nature and thecomplex linkages between residues have made glycan analysis a challenging task,and thestrong hydrophilicity of glycans which leads to poor ionization efficiency and sensitivity inmass spectrometry (MS) largely impedes the N-linked glycan identification and structureanalysis by MS.To solve the problem, in this thesis, we devoted to develop the novel chemicalderivatization strategy for N-glycan analysis by MS. Based on the amination reaction toreducing end of carbohydrate,30kinds of commercial chemical reagents were selected andscreened for their hydrophobic, alkaline, reactive group and ion carrying capacity.Consequently, two highly hydrophobic polycyclic aromatic hydrocarbons,1-pyrene butyrichydrazide (PBH) and ethidium bromide (EB) were selected for glycans derivatization fortheir strong hydrophobic property. Standard oligosaccharide and N-linked glycans obtainedfrom standard glycoprotein were used to evaluate the derivatization efficiency and MSdetection sensitivity. Under the optimized condition, the derivatization efficiency of thesetwo derivatizing reagents is above98%for either PBH or EB. About13and17-foldenhancement in signal intensity of the glycans in matrix assisted laser desorptionionization/time of flight mass spectrometry (MALDI-TOFMS) were obtained after PBH orEB derivatization. The limited detection of PBH or EB derivatized maltoheptaose reaches8fmol or6fmol in MALDI-TOFMS, respectively. For N-linked glycans from glycoproteins,PBH or EB derivatization leads to an obvious increase in the number of identified glycans.Furthermore, EB derivatization is particularly effective in improving the detection ofsialyated glycans by introducing a fixed cationic charge to the derivatized glycan. Based on the promising studies above, we further synthesized two new chemical reagents(4-Hydrazinyl-4-oxobutyl)triphenylphosphonium (TPP-PrG) and bromideNα-Benzoyl-L-arginine hydrazide(BRh). Standard oligosaccharide DP7was used toevaluate the derivatization efficiency and detection sensitivity of these two new reagents.About70and35-fold enhancement in signal intensity of the glycans in MALDI-TOFMSwere obtained after TPP-PrG or BRh derivatization.Disease development and progression are usually associated with alternations inglycosylation on serum proteins. Glycans released from serum proteins hence provide avaluable source of biomarkers. To further probe the application feasibility of the newdeveloped derivatization strategies in a real bio-sample, N-linked glycans obtained fromglycoprotein of serum from health people were used to evaluate the derivatizationefficiency and detection sensitivity with TPP-PrG and BRh respectively. Isoniazid (INH)was selected to be the control for the comparison analysis. The constitutions of serumporteins are very complicated with the12or greater orders of magnitude in proteinconcentration. The ability of mass spectrometry to effectively monitor the low abundantspecies is limited. The intrinsic large dynamic range of serum proteins makes the proteomicanalysis very challenging. Serum proteins were first pre-separated to remove the highabundant proteins, so that the low abundance proteins can be enriched and further reducethe interference of N-glycans from high abundance proteins to N-glycans identificationfrom low abundance proteins. Then the enriched glycoprotein was digested with PNGase Fenzyme to release the N-glycans and applied the two new synthesized reagents forderivatization of N-glycans mixture and further analysis by mass spectrometry.Consequencely, more than10N-glycans including51glycan types from human serum weredetected by the two new derivatization reagents compared to the classic reagent INH withthe MALDI-TOFMS analysis. There are total81N-glycans including137glycan typeswere detected by these three derivatization reagents in common, in which,20kinds ofN-glycans were only detected by the newly developed reagents. Only117N-glycans weredetected by the classic fluorescent labeling8-Aminopyrene-1,3,6-trisulfonic acid, trisodiumsalt (APTS). Therefore, the two newly developed derivatization strategies provide thevaluable tools for low abundance glycan identification or structural analysis in complex samples by mass spectrometry. This strategy improved the detection efficiency of N-glycansby MALDI-TOFMS, which made a technical support for the carbohydrate biomarkersdiscovery.Overall, in this thesis, a multifaceted evaluation of the commercialized derivative reagentswith different structure was first performed. The improvement of basic and hydrophobic,and introduction of charge by the derivatization were found to be advantageous to enhancethe signal strength of the glycans in mass spectrometry detection. Based on the discovery,two polycyclic aromatic hydrocarbons derivatization reagent EB and PBH were selectedand evaluated in N-glycan detection by MS. Both reagents could significant increase thesignal intensities for17and13-fold in MS and MS/MS, respectively. Furthermore, thestrong hydrophobic and cationic nature of EB leads to evidently improved identification ofglycans with sialic acid terminals, which is particularly useful in glycan analysis in realsamples. This work also reports the facile and sensitive derivatization approach foranalyzing N-linked glycans by developing two new synthesized derivatization reagentsTPP-PrG and BRh. Both derivatizing reagents could obviously enhance the signal intensityand improve the fragmentation behavior of glycans in mass spectrometry analysis. Usingthe modified derivatization conditions, derivatization with TPP-PrG and BRh could proceedcompletely under mild conditions with fewer sample handling and reduced sample loss. A70or35times increase in signal sensitivity was obtained by TPP-PrG and BRhderivatization respectively, and obviously improved S/N was also achieved for bothstandard oligosaccharides and N-linked glycans released from glycoprotein. The the newderivatization strategies were used in the investigation of the N-glycans profiling of healthyhuman serum, and52N-glycans including92glycan types and51N-glycans including95glycan types were detected by labeled with TPP-PrG and BRh, respectively, indicated thatthese new developed strategies provide a powerful tool for N-glycans profiling analysis ofcomplex and low abundance samples by mass spectrometry.