Rapid Enzymatic Digestion Of Proteins And Single-cell Proteomic Analysis Methods

Through the qualitative and quantitative analysis of protein molecules that realize functional activities in organisms,proteomics technology helps people to deeply understand the molecular mechanism of biological processes,and provides an important basis for the discovery of biomarkers,clinical diagnosis,pathological analysis and drug treatment.Proteomic analysis at the single-cell level can interpret information related to tissue microenvironment and cellular heterogeneity,which is of great significance for studies on embryonic development,tumor typing,cancer metastasis,and rare cell analysis.Due to the sparse and complex protein content of single cells,the bottom-up shotgun proteomics technology based on biological mass spectrometry shows unique advantages.The sample preparation process of shotgun proteomics technology is complicated,and the overnight protein enzymatic reaction step restricts the efficiency of sample preparation.At present,the identification depth and throughput of single-cell proteomic analysis techniques are difficult to meet the needs of practical biomedical research.The purpose of this work is to explore methods suitable for micro-sample systems that can significantly promote protein enzymatic hydrolysis,improve the efficiency of shotgun proteomics sample preparation,use microfluidic technology and liquid chromatography-mass spectrometry(LC-MS)system to establish a complete workflow of single cell proteomic research with high sensitivity and deep coverage.In Chapter 1,we introduce the reported rapid protein enzymolysis technologies from three categories: introducing energy,using microsystems,and changing the form of enzymes.According to the different sizes of reaction systems,we introduce the reported single-cell proteomic analysis methods based on the bottom-up technology.In Chapter 2,we designed and built fast protein enzymatic hydrolysis systems based on infrared laser,microwave and ultrasonic energy sources,using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry(MALDI-TOF MS)and electron spray ionization mass spectrometry(ESI-MS)to detect digested peptides and evaluate the enzymatic digestion efficiency.Experiments show that these three energies can promote the enzymatic digestion rate of proteins.In the infrared-assisted protein enzymatic hydrolysis system,the proteins in the droplet could obtain a good reaction result after being irradiated by a 150 m W 808 nm infrared laser for 165 s.The reaction effect of large-volume samples was better when the ultrasonic wave was applied for 10 min,but the disturbance of the micro-droplet system was too large.The result of microwave was better than that of infrared laser,which was more suitable for the treatment of large-scale micro-droplet samples than ultrasonic wave.The protein in the micro-droplet was heated by microwave for 30 s to obtain better enzymatic hydrolysis result.The energy-based rapid digestion methods could achieve the same protein sequence coverage as the overnight digestion in 1 min,but the peptide signal intensity could only reach 20% of the overnight digestion.It showed that the energy-assisted enzymatic hydrolysis method has the effect of promoting enzymatic hydrolysis but could not completely replace the overnight enzymatic hydrolysis reaction.In Chapter 3,we develop a bottom-up single-cell proteomics preprocessing workflow based on polytetrafluoroethylene(PTFE)chip and sequential operation droplet array(SODA)technology.SODA technology could achieve accurate picking of single cells and flexible manipulation of nanoliter reaction solution.PTFE chip and anti-evaporation device controled the pretreatment reaction volume to keep 150-300 n L,the nanoliter liquid chromatography,self-made capillary chromatographic packed columns and high-resolution mass spectrometer was used to complete the high-sensitivity analysis and detection of single-cell proteomics samples.We optimized the gradient time,flow rate and spray voltage of liquid chromatography with 200 pg He La cell extraction protein enzymatic hydrolysis standard as the sample,and obtained the separation and analysis strategy for single-cell samples.The relative standard deviation(RSD)of the standard sample was less than 2%.We performed proteomic analysis on single He La cells,identifying an average of 558 proteomes from eight of single He La cells in DDA acquisition mode,with the highest group of single cells identifying 921 proteomes.We performed kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis on the obtained data,and the results show that the identified proteomes can enrich dozens of pathways,involving various biological processes such as metabolism,synthesis,circulation,and diseases,demonstrating the application prospects of single-cell proteomic analysis in the field of biomedicine.