Multidimensional Liquid Chromatography/Mass Spectrometry Analysis System And Related Theory

Multidimensional liquid chromatography separation system is a type of chromatographic technique that enhances peak capacity by combining different chromatography separation modes.It plays an important role in separation,analysis,purification and preparation of complex sample.This paper investigated how to establish a multidimensional liquid chromatography / mass spectrometry analysis system and developed related theories systematically based on different aspects with a balance of theoretical and practical perspectives.On the basis of the aforementioned research,a multiple novel two-dimensional liquid chromatography systems was built for sample analysis and preparation and software and control systems related to chromatography separation and mass spectrometry detection were developed.To explore new technologies and novel methods for proteomic research,a full twodimensional microcolumn liquid chromatography-electrospray ionization time of flight mass spectrometry platform was built for online analysis of proteins by coupling ion exchange chromatography and reversed-phase chromatography together with an interface using an automatic-switched ten-port valve with two C8 trap columns.Using this twodimensional separation system,up to twice as many peptides with unique m/z were detected than one-dimensional separation system under the same conditions.Peak capacity up to 800 was achieved while using this system to separate proteolytic breakdown products of low metastatic mouse hepatocellular carcinoma cells.A novel integrated protein pretreatment system was designed and built,which enables continuous online proteomic research.18 differential membrane proteins were identified through analysis of membrane proteome by two different protein digestion methods,14 and 4 of which were quantified by trypsin and pepsin digestion methods respectively,showing good complementarity.Meanwhile,multidimensional liquid chromatography separation system consisting of different chromatography separation modes offers advantages in preparation of single or multiple components from a complex sample.Establishment of a theoretical model of solute transport processes in chromatography column provides the basis for analysis of spectral features with very large volume injection in preparation chromatography and creating a three-phase two-dimensional preparation liquid chromatography system consisting of three chromatography separation columns with three-port valve and computer control software adopted,addressing conflicts between large volume injection and high separation efficiency by maintaining a good balance.By assembly of multiple three-phase chromatography systems,a quasi-continuous two-dimensional preparation chromatography system was built,enabling automatic continuous injection,separation and purification of sample with software controlling.Parallel columns of the same or different separation modes can be used on demand in the second dimensional separation system for concurrent preparation of multiple target compounds.A continuous chromatography system for preparation was built and applied to continuous separation of total flavonoids and chlorogenic acids from water extracts of lonicera japonica.Separation of total flavonoids and other components was achieved once with purity of total flavonoids up to 90.3%.It is crucial to investigate analysis conditions of mass spectrometry which is the most important detector of high performance liquid chromatography.Studying of effects of differences of retention times of isotope sample H/D on MS/MS quantification results illustrated that 2,4 deuterium-labeled sample has little effects on MS/MS quantification results.As the advance of researches about HPLC/MS methods for analysis of complex samples such as proteomic sample and natural products,large amount of data is available.To better understand these data,extraction of valuable information and exploration of theories to explain patterns are required.A crowdsourcing-based natural product database was created,ensuring its growth.A novel scoring algorithm which is optimized for IPTL experiments was designed: All Ions Scoring Algorithm(AISA).The identification algorithm can be easily extended to have quantification functions.And the coverage of quantification can be improved.A selected reaction monitor data processing pipeline was developed to automatically analyze large amount data.By selecting precursor and product ions,sensitivity and accuracy can be significantly improved in selected reaction monitor experiments.The method is of high selectivity,well reproducible,highly sensitive and of wide dynamic range.