Quantitative Phosphorylation Proteomics Of Nonfunctional Pituitary Adenomas

BackgroundPituitary adenomas(PitNETs)are common neuroendocrine tumors derived from adenohypophysial cells.Clinically,PitNETs can be divided into functional pituitary adenomas(F-PitNETs)and non-functional pituitary adenomas(NF-PitNETs)according to blood hormone levels.Compared with F-PitNETs,the corresponding hormones in blood of NFPitNETs do not increase significantly,which increase the difficulty of early diagnosis.The pathogenesis of NF-PitNETs is a complex bioprocess which involved in many genes,factors and processes.Phosphorylation is an important post-translational modification(PTMs),and abnormal phosphorylation is always related to tumor mechanisms.Tandem mass tag(TMT)labeling,TiO2 phosphorylated peptide enrichment combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)technology were applied to identify the differentially phosphorylated proteins(DPPs)and phosphorylated sites between NF-PitNETs and control tissues.It is necessary to study the related biological pathways and functions from proteomic level,so as to understand the pathogenesis of NF-PitNETs.It provides a new theoretical support to explain the molecular mechanism of NF-PitNETs.Furthermore,it has significance for the prediction,prevention,personal medication(PPPM),and discovery of drugs and therapeutic targets of NF-PitNETs.Method(1)In order to obtain the differential phosphorylated proteins(DPPs)and phosphorylated sites(p<0.05)comparing non-functional pituitary adenomas(NF-PitNETs)with normal pituitary glands,the main steps include:proteolysis,peptide concentration determination,tandem mass tag(TMT)reagent labeled peptide and equal mixing,the mixed peptide was enriched by titanium dioxide(TiO2),liquid chromatography-tandem mass spectrometry(LCMS/MS)analysis as well as data analysis.(2)To understand the biological roles of related pathways in the pathogenesis of NF-PitNETs,KEGG pathway analysis from DAVID database was applied to identify the signaling pathway network changes in DPPs.(3)Enrichment analysis was conducted for functional characteristics of DPPs by GO analysis from DAVID database,including BP(biological Process,such as participating in cellular immunity,apoptosis,cell proliferation,etc.),CC(cellular component studies the location of a protein in a cell,such as in the membrane,mitochondria or nucleus,etc.),MF(molecular function,such as the name of an enzyme or carrier protein,etc.),Where the differential genes are found to cluster in each of the three categories gives an overview of which processes or functions are likely to be crucial in cancer.(4)Kinase prediction of DPPs via PhosphoSitePlus database is conducted to search for related substrates and predict relevant biomarkers and therapeutic targets.(5)Immunoprecipitation(IP),western blotting(WB)were used to verify the differences of DPPs from quantitative phosphoproteomics based on LC-MS/MS.Results:(1)Differentially phosphorylated proteins(DPPs)in NF-PitNETs.In total,595 DPPs with 1412 phosphosites were identified in NFPitNETs compared to controls(p<0.05).506 DPPs with increased phosphorylation levels including 4 DPPs(FGA,CDS2,DNM1,and SRRM1)that were only phosphorylated in NF-PitNETs but not in controls.(2)KEGG pathway enrichment analysis of 595 DPPs with 1412 phosphosites identified nine significant signaling pathways(p<0.05),including spliceosome pathway,the RNA transport pathway,the proteoglycans in cancer,SNARE in vesicular transport,platelet activation,bacterial invasion of epithelial cells,tight junctions,vascular contraction,endoplasmic reticulum protein processing.(3)GO analysis was used to reveal the functional characteristics of DPPs in NF-PitNETs.(ⅰ)For BP analysis,595 DPPs were significantly assigned to 122 BPs,which were mainly involved in RNA processing(mRNA processing,RNA splicing,RNA export,and transcription),the regulation of subcellular organelles(Golgi vesicle,endoplasmic reticulum/ER processing,and chondriosome regulation),cell-cell interaction(cell adhesion,and cell division),and cellular reactions to specific matter.(4)The PhosphoSitePlus database is used to analyze the kinases for the identified phosphoproteins in NF-PitNETs and controls,which identified seven kinases,including GRP78,WSTF,PKN2,PRP4,LOK,NEK1,and AMPKA1.(5)Calnexin(p=5.32×10-7)is randomly selected from 595 DPPs of NFPitNETs to verify differences in phosphorylation between NFPitNETs and controls.The expression differences of phosphorylation sites is identified by LC-MS/MS quantitative phosphorylation proteomics,S554(T/N=10.67,p=7.88×10-6),T562(T/N=15.45,p=1.07×10-5),S564(T/N=6.88,p=1.03×10-5)and S583(T/N=10.48,p=5.32×10-7).IP combined with WB analysis showed that compared with the control pituitary tissue,calcinin was highly expressed in NF-PitNETs and the overall phosphorylation level of NF-PitNETs was higher than that of the control.These results are consistent with quantitative phosphoproteomics based on LC-MS/MS.ConclusionTMT-based quantitative proteomics coupled with TiO2 enrichment of phosphopeptides effectively identified and quantified protein phosphorylation at residues of Ser(S),Tyr(Y),and Thr(T)in human NFPitNETs compared to controls.This study provided the first quantitative phosphoproteomic profiling of human NF-PitNETs relative to control pituitaries,and phosphorylation-mediated biological processes and molecular pathway network changes in NF-PitNETs.These findings add new information on the roles of phosphorylation in PitNETs and provide new insight into elucidating the molecular mechanisms of NF-PitNETs,exploring significant therapeutic targets,and discovering new biomarkers for the effective management of NF-PitNETs.