Identification And Validation Of Glycoprotein Biomarkers In PD And Its Progression

PD is traditionally considered a movement disorder that results from a relatively selective loss of neurons in the brainstem,including dopaminergic neurons in the substantia nigra pars compacta,with subsequent loss of striatal dopamine and accompanied by the formation of intraneuronal inclusions called Lewy bodies that containα-synuclein as one of the major proteins.PD is the forth common disease occured in senior citizens.Although some unique proteins,such asα-synuclein and DJ-1, were studied,the pathogenesis of PD is still unknown.Additionally,selection of biomarkers that can predict and examine PD and its progression is important as well.Protein glycosylation regulates protein function and cellular distribution.Furthermore, aberrant protein glycosylations have been recognized to play major roles in human disorders,including PD and AD.Glycoproteomics,a branch of proteomics that catalogs and quantifies glycoproteins,provides a powerful means to systematically profile the glycopeptides or glycoproteins of a complex mixture,and therefore,cany great potential to be diagnostic and/or prognostic biomarkers.Until recently,very little has been known about the role of glycosylated proteins in PD.Application of this mass spectrometry-based technology will improve the identification and selection of glycoprotein biomarkers in PD and its progression.In this study,hydrazide resin was used to enrich glycoproteins which were isolated from brain tissue and CSF of normal people and PD patients.To perform quantitative analysis of glycoproteins unique to PD and PD progression,samples were digested with trypsin,followed with iTRAQ labeling.With MALDI-TOF/TOF,the glycoproteins/glycopeptides spectra were extracted and searched against the IPI human protein database with ProteinPilot^TM software with the Paragon^TM method.The raw peptide identification results flom the Paragon^TM Algorithm(Applied Biosystems) searches were further processed with the Pro Group^TM Algorithm within the ProteinPilot^TM software before final display.The Pro Group Algorithm uses the peptide identification results to determine the minimal set of proteins that can be reported for a given protein confidence threshold.The MS/MS analysis revealed a total of 394 non-redundant glycoproteins in human brain tissue and 283 non-redundant glycoproteins in hurnan CSF.In comparison with the existing publicly accessible database,343 of human tissue glycoproteins and 243 of human CSF glycoproteins were annotated in UniProtKB/Swiss-Prot and the ISB database as glycoproteins with known glycosylation sites or probable/potential glycosylation sites.The specificity of this approach were 87%(343/394) and 86% (243/283) separately.All reported data were based on 95%confidence for protein identification as determined by ProteinPilot(ProtScore≥1.3).Additionally.when the glycoproteins identified in human brain tissues and CSF were classified by GO analysis,it was apparent that a majority of the proteins belong to either the extracellular compartment or are associated with the plasma membrane.This is entirely consistent with the claim that most membrane proteins are glycosylated,and that a significant portion of glycoproteins are designated for secretion into the extracellular fluid and thereby enter blood or CSF.In this study,98 proteins were seen in brain tissue(a total of 394 proteins) and CSF(a total of 283 proteins) to account for 25% of brain tissue glycoproteins and 40%of CSF glycoproteins,besides 43 proteins are associated with CNS.These overlapping proteins identified with glycoprotein isolation are more likely related to PD pathogenesis.With quantitative analysis,more than 40 glycoproteins were changed in normal brain tissue and PD patients' brain tissue,which indicates that these glycoproteins might be the potential biomarkers in PD and its progress.For further study,the total plasma glycoproteins were isolated from normal people,PD in different stage and AD patiens by hydrazide resin to perform western blotting analysis.The result shows that 6 unique glycoproteins have notable expression both in group samples and in sigle samples.Thus, these candidate glycoproteins might be the potential biomarker in PD and its progression.It should be emphasized that this dataset represents the first systematic analysis of glycoproteins in human brain in normal and diseased settings.This study identificated and selected several glycoproteins as potential biomarker in PD and its progress,which provided important imformation and evidence to illustrate PD pathogenesis.