| Research on proteomic aims to reveal the phenomena and regular patterns of life at molecular level,which has become a main breakthrough and the forward position of life science and biotechnology in the 21st century.At present,the development of proteomic are facing two main challenges regarding the technical bottleneck of compatibility between traditional methods and modern instruments,as well as the improvements of identification depth and qualification accuracy of significant biological samples by advanced technologies.Based on the above research gaps,this topic focused on two works,one of which is the development of parallel channel multidimensional protein identification technology(PC-MudPIT)and the other is the quantitative research of data independent acquisition(DIA)technique on urine proteomic.Multidimensional Protein Identification Technology(MudPIT),developed by John Yates at Scripps Research Institute in the United States 20 years ago,showed incomparable advantages in deep proteomic identification of highly complex samples because of its direct on-line automatic fraction and analysis.However,a major limitation in such classic MudPIT system was the incompatibility with the improving sensitivity and ultra-high performance of modern mass spectrometers in recent years,because MudPIT introduced different concentrations of salt into the mass spectrometer during the elution of peptides along strong cation exchange(SCX).Although multiform trap column can be introduced to make some improvements,realized desalination will increase the dead volume,destroy the continuity of the reversed phase separation dimension,as well as decrease the chromatographic resolution and sensitivity.Gradually,the fractionation process will go back to the original off-line operation mode.Therefore,it is very important to break through the traditional method to solve the technical bottleneck of the classic MudPIT regarding its compatibility with modern instruments.In order to solve the above problem and bring MudPIT back to the frontage of proteomics research,our first part research focused on technological development of PC-MudPIT.We innovatively designed two parallel channels that can operate simultaneously and independently,and finally achieved direct desalination without adding trap column.This new design of two parallel channels replaces the two-phase trap column in the classic MudPIT.One channel is working on separation and desalting process,while the other is executing second dimensional peptide analysis.In addition,due to the characteristics of parallel alternating operation,there did not exist time gaps during the whole experimental process,thus further improved the efficiency and throughput,and made online phosphoproteome analysis possible as well.Recently,DIA technology has gradually been applied to the frontier proteomics research with high coverage,reproducibility and accuracy,which is the result of fragmentation of ions is independent to the composition of precursor ions under data independent acquisition mode.However,up to now,there are still several challenges in the application of DIA technology in complex matrix samples.Particularly,the identification depth of DIA technology for urine samples remains at the level of 1,000-2,000,which leads to the non-invasive obtained urine sample cannot be an ideal material for clinical disease research.Thus,it is especially crucial to improve the identification depth and quantitative accuracy of protein among important biological samples(such as urine)by using advanced technology.In the second part of the study,for the first time,we set up an optimized DIA method based on advanced timsTOF pro mass spectrometer(Bruker,Bremen,Germany),for comprehensive mapping of the urinary proteome by optimizing different parameters of LC-MS.Through this method,the average identification depth of urine protein group increased from less than 2,000 to 4,000 protein per sample,which is a breakthrough in solving the problem of low coverage depth of urine protein group.We applied this combination technique to the urine and serum proteomics studies of patients with COVID-19 to reveal the multifaceted effects of this newly emerged disease on human body.We found that COVID-19 patients presented immunosuppression and impaired tight junctions in the early stage of infection and some degree of immune activation in the later stages,which might lend evidence to a two-stage model of COVID-19. |
