Structural Analysis Of Oligosaccharides In A Complex System And Development Of Phosphorus-Containing Reagents For Saccharides Mass Spectrometry Signals Enhancement

Carbohydrates mediate many important biological processes in vivo.The structural analysis of carbohydrates thereof significant and drawing extensive attention.However,bearing the most structurally diverse nature,as well as their highly polar and lack of chromophores,carbohydrates are very unfriendly for researchers."Speedy,Specific,requiring Small amount of analyte" is the precise description of mass spectrometry.The cutting-edge mass spectrometers have become the most utilizing detectors for glycomics.Equipped with separation tools and tandem mass analyzers,mass spectrometers are ongoing successes on structural and quantitative analysis of carbohydrates.Nonetheless,the poor ionization efficiencies of glycans hinder the directly measurement by mass spectrometry.Derivatization of glycans is often needed to improve the stability,ionization efficiency,and quantitation accuracy for glycomics analysis.The derivatization reagent commonly can add a chromophore and/or mass reporter to the glycan,as well as lowering the polarity of the glycan.making it possible to perform in-depth profiling.This thesis focusses on two topics:the mechanism of unprotected monosaccharides oligomerization under the induce of phosphorus reagent:the development of mass spectrometry signal enhancement reagent for carbohydrates derivatization.Both of the topics were related to the carbohydrates'derivatization strategy.In this thesis,monosaccharides oligomerized under the inducing of penta-coordinated phosphorus reagent.Disaccharides from the synthetic oligosaccharides'library,which bearing the minimize degree of oligomerization,were separated in peracetylated form.The peracetylated disaccharides sample was further profiled by nuclear magnetic resonance and high-performance liquid chromatography coupled with mass spectrometry.It was found that the main glycosidic bonding pattern is(1?6)linkage with 66%regioselectivity for each step.The ratio of ?-(1?6)and ?-(1?6)glycosidic bonds formed is around 1:1.The reactivity of 1-OH group from aldoses and the driving force from penta-coordinated phosphorus intermediates are the two critical factors for the oligomerization.On basis of the mass spectrometry signal enhancement of amino acids derivatized by phosphinyl group,and the mass spectrometry signal enhancement of carbohydrates derivatized by hydrazinyl group,this thesis planned to develop two derivatization reagents,named 4-DPPPHN and P4HZD.Unfortunately,the phosphinyl substituted reagent 4-DPPPHN couldn't be prepared in pure form and was very unstable.The roughly prepared 4-DPPPHN did not show good signal enhancement to glycans,either.On the other hand,P4HZD,the phosphonium salt containing permanent charged hydrazide derivatization reagent showed great stability.P4HZD can amplify the signal intensities of oligosaccharides from maltodextrin which DP?7 to 25-120-fold in MALDI-MS.Maltopentaose also gains 20-fold S/N ratio enhancement after derivatization by P4HZD in MALDI-MS.Based on quantitatively measurement in HPLC-ESI-MS,P4HZD has 80-90%labeling efficiency to glucose and maltopentaose,as well as 4-6-fold signal intensity amplification for maltopentaose and 688-fold for glucose.In the case of natural N-glycans released from RNase B,P4HZD derivatization can acquire 100-fold signal intensity amplification and 40-fold S/N ratio enhancement.The unique feature of P4HZD signal enhancement to monosaccharides portended its huge potential on the research of central carbon metabolism and imaging mass spectrometry.