Identification And Quantification Of Homologous Pirin Proteins By MS

Pirin proteins belong to the Cupin family with functional diversity.It is a highly conserved metal nucleoprotein that is widely expressed in eukaryotes and prokaryotes.It is recently reported in the literature Pirin is highly expressed on melanoma,and the difference in protein expression is related to different types of malignant tumors.However,there is no data now available of Pirin proteins distribution in the relevant tissues and cells.Therefore,it is very important to establish an analytical method that can quickly identify and accurately quantify homologous Pirin proteins in biological samples.The multi-reaction monitoring mass spectrometry method(MRM-MS),due to its high sensitivity,high selectivity,and multiplexed quantification capability,has shown special advantages in the analysis of low-content samples of complex biological matrices.In this paper,two homologous Pirin proteins with quercetinase activity(human h Pirin and Escherichia coli Yhh W)whose crystal structures were successfully expressed and resolved by our group were selected as the research object.The respective marker peptides were screened out,and a MRM-MS-based method was established,which was applied to the identification and quantification of h Pirin and Yhh W in a variety of biological samples(strains,hepatocyte,tissues,melanoma cells).The work mainly includes the following three aspects:(1)Develop a MRM-MS method based on marker peptides to determine the expression of different fragments of Yhh W.The Yhh W protein was expressed in E.coli,cleaved with trypsin,and the marker peptides were screened by Blast analysis.An identification and accurate quantification of Yhh W by liquid chromatography-mass spectrometry(quantification limit of fmol level,RSD is less than 13%,accuracy is 97%-107%),can be used for the determination of Yhh W content in different E.coli categories.(2)Construct a dimethyl-derivatization based MRM-MS method to identify h Pirin in hepatocytes.The h Pirin protein was expressed heterologously in E.coli,cleaved with trypsin,and the marker peptides were screened by Blast analysis;the tryptic products were dimethylated and isotope labeled with formaldehyde and sodium cyanoborohydride to improve both the chromatographic retention and the ionization efficiency(the sensitivity is generally increased by more than 5 times,and the detection limit reaches fmol level),which can be used to identify h Pirin in hepatocytes.(3)Compare the Pirin expressions in different biological samples.By comparing the sequence of different types of homologous Pirin proteins to identify the same marker peptides.The different tissues distribution of Pirin protein in pork was identified;and the differences of h Pirin expression in melanoma cells treated with different concentrations of flavonoids(taxifolin and apigenin)were measured.It was found that taxifolin and apigenin can inhibit the proliferation of A375 melanoma cells and cause differences in Pirin expression.