Proteomic Analysis Of Prolactinoma By Immuno-laser Capture Microdissection

The pituitary is a significant endocrine gland responsible for the regulation ofvarious physiologic and metabolic processes.Pituitary adenomas comprise nearly16.7% of intracranial neoplasms,making it the third most common intracranial tumorafter gliomas and meningiomas and prolactinomas account for 25-14% of the wholepituitary adenomas.Although most are pathologically benign and grow slowly,prolactinoma presents many symptoms in patients:amenorrhea and galactorrhea,dysgenesia in female patients and infertility and eractile dysfunction in male.A smallnumber of prolactinomas belie their invasive histology by undergoing perisellarinvasion,postoperative recurrence,and,on rare occasions,craniospinal and systemicspread.Increasing evidence suggest that characterization of DNA and RNA alone willnot be sufficient to elucidate mechanisms of disease and diagnostic markers.Humanpituitary adenoma proteomics would offer an efficient means for a comprehensiveanalysis of pituitary protein expression.Currently,the main technique for high throughout Proteomics research istwo-dimensionalGel electrophoresis,(2-DE) 2-DE technique can separate Proteins bydifferent characters of the proteins.Several two-dimensional electrophoresis basedproteomic pituitary adenoma researches have been reported,while only fewdifferential expressed proteins were identified and can not presented thehigh-throughout of the Proteomics technique and all previous research had a commonshortcoming in sample preparation-heterogeneity.Proteomic analysis techniques arethe most accurate measurement when applied to homogenous cell populations.However,the significant admixture of variable non-neoplasmic cells wouldcompromise the application of protein extracted from bulk tumor tissue and make itvery difficult to discover the significantly expressing protein hidden in specificcell population.Laser capture microdissection (LCM) from stained tissue sectionsallows the isolation of morphologically identified cell populations down to thesingle-cell level for proteomic analysis,and thus can ameliorate the problem of tissueheterogeneity.The sample loading in these new techniques was smaller than 2-DE,while theseparation efficient was higher.Considering the shortcomings of 2-DE and theprogression of Proteomics technology,we decided to apply tandem-mass spectrometershotgun method,namely nano scale high performance two-dimensional liquid chromatography combing with immuno-laser capture microdissection to directlyidentify the Proteins from mixture of tryptic peptides.This dissertation consists of 4 parts and contents are summarized as follows:In the first chapter, the status quo of ion source and mass analyzerand the technology developments in proteomics such as multi- dimensionalanalysis of protein identification technology(MudPIT),Tissue Imaging-MS,Lasercapture microdissection (LCM),and quantitative methodologies,were summarized.In the second chapter the research focused on the proteomic profile of prolactinomaand the normal pituitary by immuno-laser capture microdissection.We compared thedensity of positive prolactin cells in immunohistochemistry of frozen normal andprolactinoma before and after pretreatment with different concentration and durationto find an optimal method for LCM to prepare proteomic samples.There are vastarchives of formalin-fixed tissues spannin allowing acquisition of the necessarynumbers of samples to carry out biomarker discovery study.Because the formalinfixation process resulted in the cross-linking of proteins,and thus,intact proteinscannot be efficiently extracted.In this study,2% SDS were used to extract proteinsfrom formalin-fixed prolactinoma for shotgun proteome analysis.So the next step,weharvested the prolactin cells from normal pituitary and prolactinoma (frozen andformalin fixed) from 50000-10000 by Acturus LCM under the direction ofimmunohistochemistry and extract the proteins by Lysis buffer containing urea (7 M)and thiourea (2M) or 2% SDS and the digestion is enough for the shotgunLTQ-Qrbitrap mass spectrometry analysis.Protein expression Profiles of prolactincells from normal pituitary and prolactinoma were identified by thermo Finniganlinear ion trap tandem mass spectrometer and liquid chromatography.We found moreintense and specific staining could be obtained when sections were pretreated with0.2% Triton X-100 for 4 min.A prolactinoma proteome consist of 2243 proteins wasobtained.Categorization of the proteome revealed an access to various proteins withdiverse characteristics,including pituitary hormones,cellular signals,biosyntheticprocess,cellular metabolic process,cellular localization proteins,response to stressproteins,transport proteins,even some low abundance phoshpoproteins.It was foundthat incubation of formalin-fixed tissue in a lysis buffer containing 2% SDS at hightemperature for 30 min led to an optimal protein yield and the nearly identical numberof proteins (1521) and characters such as molecular weight (MW)and isoelectric point(PI) were compared with those identified from frozen-fresh tissue(1652).At the last part of this chapter we present the isolation of prolactin cells in postmortem normalpituitary using the immuno- LCM approach,and the systematic identification ofextracted proteins by LC-MS/MS.Together,more than 1600 proteins were detected inthe LCM samples.All the proteomic profile studies provide a foundation for thefurther tumorgenesis of prolactinoma.Understanding the differential protein expression will help to develop newprevention and treatment methods for prolactinoma.In the third chapter theimmuno-LCMed prolactin cells were used in this study to provide a pool samplesfrom 8 prolactinomas and 8 normal pituitaries.Quantitative proteomic profile ofcontrol group was compared with that of prolactinoma group to search for differentialexpressed Proteins.Identification of Proteins was achieved using Protein Pilotsoftware and searched against the International Protein Index (IPI,V 3.45) proteindatabase.Relative quantification of proteins using iTRAQ technology combining withnano scale 2D LC-MS/MS(QSTAR),was employed to establish the prolactin cellproteome (263proteins) and obtain 82 differential protein expression inprolactinoma.The results may provide the insights into the mechanism of theprolactinoma.5 differentially expressed Proteins were selected to validation bywestern-blot and the results and statistics analysis indicated a consistent proteinexpression change,iTRAQ^TM technology combing with LCM and nano scale 2DHPLC-MS/MS was successfully applied to analysis the differential proteins betweenprolactinomas and normal pituitary to investigate the mechanism of tumorigenesis inprolactinoma.In the fourth chapter the identified differential proteins were analysis bybioinformatics with DAVID (The Database for Annotation,Visualization andIntegrated Discovery) and KEGG (Kyoto Encyclopedia of Genes and Genomes).Analysis result indicated that the identified differential proteins mainly in intracellularpart and had a wide molecular function.KEGG analysis indicated the differentialproteins had a close correlation with focal adhesion pathway and regulation of actinskeleton.The bioinformatics analysis will provide a new perspective to elucidate thetumorigenesis in prolactinoma.