| The types and quantity of proteins in biological samples are vital in biomedical researches.Mass spectrometry-based proteomics analysis can reveal potential biomarker of various diseases.However,there are some challenges in this field.The low amount of proteins in the clinical samples and in the single cell both require much lower loss rate when preparing samples.Compared to mass spectrometry,the technology of sample preparation is under developing.The steps of tranditional methods for sample preparation includes reduction,alkylation,digestion,desalting and the inevitable transfer and freeze-drying in tubes.The amount of proteins that can be captured in some biological samples is limited,so they are easily lost in these steps.Monolithic column can overcome these unsolved problems.Its high porosity can provide more reaction area to enrich biomolecules to a large extent.In addition,because the monolithic columns have low back pressure and high flow rate,the precipitation of salt components can be done in the process of sample pretreatment.The functionalized monolithic columns that prepared by using one-pot method are used to pretreat microprotein to construct an integrated method for online sample preparation.Cell lysis,protein digestion and desalting are completed in monolithic column,which greatly reduces sample loss and increase the efficiency of digestion.By connecting monolithic column with mass spectrometry,we can directly analyze the samples.This improves the identification of proteins by solving the problem of low efficiency of sample transfer into mass spectrometry.1.Preparation of functional monolithic columnsButyl methacrylate(C4),dithiothreitoland,initiator and POSS reagent(MA-POSS)which contained multiple methacrylate functional groups are polymerized in one pot.The C4 functionalized monolithic column with low permeability and high poriness has better performance in enriching proteins.As the length of column increasing,the efficiency of protein enrichment also increases.The monolithic column is characterized by electron microscopy and mass spectrometry.It is found that the pore size of the monolithic column is about 1μm and the column capacity of the monolithic column is up to 50 ng protein.2.Construct an integrated method for online micro-sample preparationsBased on the monolithic column modified with functional monomer,the integrated method for online sample preparation and direct analysis is constructed.The parameters of the LC instrument were optimized,while directly analyzing peptides on the monolithic column.After 45 minutes equilibrating,the instrument has the best states to analyze samples.The proteins are digested online in different digestion conditions.It is found that 1μg/μL Trrpsin buffer can digest all the proteins within 2 hours.Compared with the centrifuge tube treatment,this method can effectively improve the number of protein identification.3.Apply and evaluate integrated method for proteomic analysis of single cellThe integrated method for sample pretreatment was applied to mouse TKR-CT26 cell repeatedly.The results were compared with the results of a large number of TKR-CT26 cells.It is found that their GRAVY scores and molecular weight distribution of TKR-CT26 cell protein are similar,which proved that the integrated method has good repeatability.Then,i RT peptides with different masses are used as internal standard to draw the linear curve of mass and peak area in MRM mode.Meanwhile,i RT peptides can be added in the cell sample as internal label.Based on its actual peak area,the loss of i RT peptides can be calculated.The average loss rate of the integrated method is 41.02%.It proves that the advantages of the integrated method has low sample loss.By setting up blank control groups,it can prove that the integrated method can cause little contamination. |
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