Establishment Of Dual Reverse Phase Chromatography Combined Parallel Reaction Monitoring For Targeted Quantitative Proteomics

Objectives The goal of this project is,firstly,Optimizing the chromatographic conditions of mass spectrometry to improve the efficiency of mass spectrometry.Seconding,through optimizing the parameter settings of PRM method,the target quantitative detection of low abundance proteins could be improved.Thirdly,to Establish the Dual Reverse Phase Chromatography combined Parallel Reaction Monitoring for targeted quantitative proteomics,which would promote the progress of the low abundance protein quantitative.Methods 1 We optimized the chromatographic conditions: increasing the inner diameter of the column and decreasing the column length;increasing the flow rate of the liquid phase elution and shortening the elution gradient,and we used the DDA method to identify the293 T extract and generate data.Then,we compared the identification number and detection efficiency between the different chromatographic conditions,at both protein and peptide levels.2 We adjusted the settings of instrument parameters which includes the isolation window of quadrupole,AGC,the Maximum Injection time of precursor,resolution of the Orbitrap,the time window of targeted peptide,and so on.After adjusting the settings we used the cell lysis from HEK293 T cells,as a positive control,to estimate and determine the best parameters for our study.3 We used pre-separated lysis from both HEK293T(0.2 ?g and 0.6 ?g peptides)and E.coli(1 ?g)to evaluate the repeatability,accuracy,and throughput of the PRM platform.Results 1 The results of MWM test shows that using 8 cm chromatographic column,we got detected 4295 proteins and 23709 peptides in 70 min,which is comparable with the results as we used 12 cm chromatographic column in 70 min gradient,4455 proteins and24369 peptides.And as we shorten the gradient to 35 mins,the number of protein detection increased twice.In this we determined the chromatographic conditions as follows: 150 ?m inner diameter 12 cm chromatographic column,elution speed 800 n L/min,and effective gradient 70 mins.2 We determined the settings of instrument parameters as follows:isolation window of quadrupole 1.5 min,AGC 1e5,the Maximum Injection time of precursor 40 ms,resolution of the Orbitrap 15000,the time window of targeted peptide1m/z.3 Using the optimized method,we quantitatively detected more the 400 peptides with low abundance in HEK293 T cell lysis.And comparing with DDA method,our novel PRM method is more accurate,with lower CV.Conclusions In the project,we optimized chromatographic condition for LC,and constructed rp-rp-LC-MS combined PRM target quantitative method.And by using this novel PRM method,we hope the efficiency and accuracy of MS detection for low abundance proteins would be improved greatly.