Quantitative Proteomics Study On The Phosphorylation Modification Of TMT Markers In Human Cell Line NB4 In Response To The Effects Of JAK2, MPL, And CALR Mutations On NAP Expression

Objective:JAK2,MPL,and CALR mutant genes are the "phenotype driving genes" of MPN.The study found that the neutrophil alkaline phosphatase(NAP)in patients with JAK2V617 F increased,while the neutrophil alkaline phosphatase(NAP)in patients with MPL and CALR decreased,the mechanism of which is unclear.In this study,a series of cutting-edge technologies such as TMT labeling,high-performance liquid chromatography fractionation technology,phosphorylated peptide enrichment technology and mass spectrometry-based quantitative proteomics technology were combined.After TMT labeling of the entire protein of CALR type1 and NB4-MSCV cell lines,quantitative omics analysis of phosphorylation modification was performed to compare the expression of phosphorylated differential proteins in the mutant gene cell line and the empty control cell line,which provide new research ideas for the mechanism study of the difference in NAP content caused by three driving genes.Methods:After constructing a NB4 cell model stably expressing JAK2V617 F,MPLW515L,CALR type1 mutant genes and control gene NB4,this study used protein extraction,pancreatic enzyme digestion,TMT labeling,high performance liquid chromatography(HPLC)classification,affinity enrichment,liquid phaseChromatography-mass spectrometry tandem analysis,database search,bioinformatics analysis to study the quantitative omics of phosphorylation in samples.Results:In this study,it was found through protein differential modification analysis that compared with NB4-MSCV,NB4-JAK2V617 F had 1434 sites with significantly up-regulated(>1.5)differential phosphorylation modification,704 with protein,1244 sites with significant down-regulation(<1/1.5),and 749 with protein;Compared with NB4-MSCV,NB4-MPLW515 L had 2405 sites with significantly up-regulated(>1.5),and 1078 with proteins,and 460 sites with significantly reduced(<1/1.5),320 with proteins;Compared with NB4-MSCV,NB4-CALR type1 had 26 sites with significantly up-regulated(>1.5)differential phosphorylation modification,24 with proteins,501 sites with significantly down-regulated(<1/1.5),and 332 with protein.According to the analysis of GO classification annotation,compared with NB4-MSCV cell line,NB4-JAK2V617 F,NB4-MPLW515 L,NB4-CALR type1 are mainly distributed in cells and organelles in the cell composition classification.In the molecular function classification,differentially expressed proteins are mainly involved in binding and catalytic activity.In the classification of biological processes,differentially expressed proteins are mainly involved in cellular processes and biological regulation.The subcellular localization annotation of the submitted protein by the subcellular localization software wolfpsort shows that NB4-JAK2V617 F,NB4-MPLW515 L,NB4-CALR type1 compared with the NB4-MSCV cell line,modified the corresponding protein of the phosphorylation site.The localization of subcellular structures is mainly distributed in the nucleus and cytoplasm.Through the enrichment analysis of KEGG pathway,it is concluded that the shared pathway of NB4-JAK2V617 F,NB4-MPLW515 L and NB4-CALR type1 is the spliceosome;the shared pathway of NB4-MPLW515 L and NB4-CALR type1 is the metabolism of fructose and mannose;The shared pathway of NB4-MPLW515 L is African trypanosomiasis,hematopoietic cell lineage,citrate cycle(TCA)and pyruvatemetabolism;the shared pathway of NB4-JAK2V617 F and NB4-CALR type1 is mismatch repair and leukocyte transendothelial migration.Among them,the differential expression of Prp5 protein in the spliceosome pathway of the three mutant gene cell lines is different.There is no significant difference in NB4-JAK2V617 F,but it is significantly downregulated in NB4-MPLW515 L and NB4-CALR type1.The annotation of GO protein classification of Prp5 indicates that the subcellular structure of Prp5 is located on the nucleus,and the biological process mainly involves RNA-related splicing,regulation and gene expression processes;in terms of cell composition,it is mainly a component element that constitutes each part of the nucleus;In terms of molecular function,it is mainly involved in ATP-related metabolism and nucleotide binding.This result further proves that there may be a certain correlation between the Prp5 protein and the difference in NAP expression of JAK2V617 F,MPLW515L,CALR type1 mutant cell lines.Conclusion:JAK2V617F,MPL,and CALR mutations can cause MPN,but there are large differences in the expression level of NAP content.The differential expression of Prp5 protein in the spliceosome pathway of each cell line is different.It may be a key protein marker that leads to differences in NAP expression in patients after mutation of three driver genes.