A Novel Accessory Of Electrospray Ionization Source For Bio-mass Spectrometry And Novel Approaches For Absolute Quantification Of Proteins

With the development of proteomic studies, deep coverage and absolute quantification of proteins are drawing more attention. Deep coverage of proteins is a precondition for discovering diagnostic markers, drug targets and critical proteins, while absolute quantification of proteins is helpful to discover and analyze the function of proteins. Deep coverage of proteins consists of the deep coverage for proteins identification and the coverage of protein sequence length. With the rapid development of liquid chromatography tandem mass spectrometry, it has become a common and efficient approach to study deep coverage and quantification of proteins, However, some problems, such as loss during sample preparation, incomplete enzyme digestion of sample and low sensitivity of mass spectrometry, disturb the identification of low abundance proteins, which restrict the deeper coverage of proteins and getting access to more quantification information of proteins. Moreover, other problems including high price of commercial reagent kits, incomplete enzyme digestion of sample, and difficulties in preparing labeled peptides, limit the development of protein quantification.To solve the above problems, in this thesis, we introduced a novel accessory of Electrospray Ionization Source for mass spectrometry to improve its sensibility; We also developed a novel enzyme digestion method to enhance the efficiency of the digestion; What’s more, two methods for absolute quantification of proteins are established:one is a novel method based on metal element chelated tags coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry, the other is a novel method using18O isotope labeled concatamers of quantification concatamer peptides (QconCAT) with the assist of reversed phase chromatographic packing combined with isotope dilution-multiple reaction monitoring mass spectrometry.This thesis consists of four chapters. In chapter1, the current methods in deep coverage and absolute quantification of proteins is summarized, and recent improvement of electrospray ionization source is reviewed. Also, influence of enzyme digestion on proteomic is analyzed. Quantification methods is introduced including18O isotope labeled and metal element chelated tags coupled with high performance liquid chromatography tandem mass spectrometry. The research contents are in the last part of chapter1in this thesis.In chapter2, with the help of an accessory of electrospray ionization source for mass spectrometry, flow rate and sensibility of Mass Spectrometry are evidently improved. With an accessory assited Electrospray Ionization source for mass spectrometry, two factors affecting the sensibility of ionization source are optimized by means of electrokinetic technology. The ionization efficiency and transmission efficiency are raised, so that the sensibility of mass spectrometry is improved. The shape of ionization source is designed through calculation software of multi-field coupling, and the function of the ionization with the accessory is examined using standard peptides. After the flow speed of ionization source comprised accessory is set at1-2ul/min, the signal increases5.6folds, signal to noise radio increases2.8folds, and the robustness is improved.In chapter3, a novel proteomic quantitation method is established based on metal element tags coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry (HPLC-SIM/MS). The labeling efficiency, the labeling stability, the chromatographic retention behavior and MS behavior of labeled peptides, linear range and accuracy of this method were examined. The results of the experiment showed that the metal element tags method has high labeling efficiency and high labeling stability, and labeled peptides with different kinds of metal tags have consistent chromatographic retention behavior and consistent mass spectrometric signal.The strategy has a high sensitivity, and its limit of quantification (LOQ) is up to1fmol. The linear range for the method is between1fmol to500fmol with R2>0.99, which means that the method has a good linearity. Moreover, this method is of high accuracy with an average recoverage of117.0%. The method has been used in the absolute quantification of a protein enolase in Thermoanaerobacter tengcongensis (TTE) with a relative standard deviation of5.7%, which indicates a high precision. In conclusion, the quantitation method is feasible and reliable for absolute quantitation of proteins. This give us an alternative for the quantitative determination of proteins in a relatively simple biological sample.In chapter4, a novel method of fast and efficient enzyme digestion for protein sample is introduced by means of increasing the amount of enzyme, and18O isotope labeled concatamers of QconCAT peptides with the assist of reversed phase chromatographic packing combined with isotope dilution-multiple reaction monitoring mass spectrometry is established. In the liquid, the radio of enzyme and protein sample is increased to1:1. It takes only two hours to achieve complete enzyme digestion of protein sample, while no self-cutting occurs. After enzyme digestion, enzyme protein and digested peptides are separated through the reversed phase chromatographic tip and the enzyme is recoverd. Moreover, enzyme digestion efficiency of the recovered enzyme hasn’t been reduced, so enzyme protein can be reused. This method is simple, and of high enzyme digestion efficiency, and the digestion time is short. Based on this, I8O labeling method is also established, with the advantage of short labelling time and no relabeling. This enzyme digestion method and18O isotope labeled method coupled with concatamers of QconCAT peptides combined with isotope dilution-multiple reaction monitoring mass spectrometry are established and are applied for absolute quantification of critical drug metablic enzymes in human liver microsomes.