The Establishment Of A Highly Efficient Peptide Prefractionation System And Its Application In Quantitative Proteome

Bottom-up mass spectrometry(MS)–based proteomics has become the most widely used approach for proteomics research.It is accomplished by first digesting a protein mixture with a protease and following with liquid chromatography-mass spectrometry sequencing of the obtained peptide digest.The fragmentation spectras are then compared with the theoretical spectras predicted from the sequences in protein databases,to pinpoint the protein sequence.However,this approach faces challenges concerning the higher complexity of the proteome sample and the wider dynamic range.In fact,to date,there is no separation method is capable of resolving all components in a single analytical dimension.Therefore,a widely accepted strategy is established to reduce the complexity of the sample by introducing Two-dimensional separation before the LC-MS/MS analysis.High pH reversed-phase fractionation,as a first off-line chromatography separation,in combination with the low pH reversed-phase fractionation in the second dimension,is increasingly used for in-depth qualitative and quantitative proteomic analysis of complex biological samples.However,the limitation for regular HPLC is the requirement for rather large amounts of starting material,and the sample loss during the tedious concatenation processes.Here we introduce Ultra Performance Liquid Chromatography(UPLC)and eight-port rotor select valve as a highly efficient and convenient way for in-depth coverage of the proteome from micro-quantity of samples.Firstly,the commonly used multidimensional chromatographic separation,the development on proteomics,as well as the progresses of quantitative proteomics are introduced.And the aim of this research and the strategies used were proposed.In order to achieve the in-depth proteome coverage,we combine the high resolution of ultra-high performance liquid chromatography(UPLC)with the automatic fractionation collection of eight-port selection valve for minimal sample requirements.This system could reach high resolution of peptide mixture while only small volume of mobile phase was collected,which minimize the sample loss.And the programmable mode of the 8-valve selector allowed the on-demand design of the number of fractions collected.The efficiency,reproducibility and stability of the system were evaluated using the HCC cell line HegG2.We observed excellent quantitative reproducibility between replicates(R2>0.95),and more than 23.52% peptide identifications over the conventional StageTip approach.The fractionation method described here is flexible,straightforward,and robust,and enable proteome analysis while with minimal sample requirements.We developed a strategy which combined the TMT reagent with the new prefractionation method to determine quantitative differences in proteomics on the tissue samples from the responding and non-responding patients after Sorafenib targeted treatment.Of the 6379 proteins,5642 proteins with accurate quantitative information,and 474 significant differential proteins were found.Among the therapeutically responding patient,238 were significantly up-regulated,235 were significantly down-regulated.Gene ontology analyses were performed to annotate the differentially regulated proteins,and the result indicated the protein involve in cell molecular regulation and signal transduction pathways.The analysis provides a very meaningful clue for the study of the molecular mechanisms of sorafenib in the treatment of hepatocellular carcinoma(HCC).Finally,we summarized the study above.The advantages,innovations and limitations of the new prefractionation method developed in this study were discussed.And the promising future of proteomics for the research and implementation of precision medicine was also prospected.