Preliminary Screening Of Depression Associated Plasma Proteins And Amino Acids: Based On Itraq

BackgroundMajor depressive disorder (MDD) is a prevalent neuropsychiatricdisorder with high disease rate, high relapse rate, high morbidity, and lowrecovery rate, which causing severe socia-economic burden. Due to theunclear pathogenesis of MDD, the diagnosis of MDD is largely based on theclinical factors and no object test is available for aiding the diagnosis.Additionally, antidepressants are the main approach of depression treatment.However, there are some limits in antidepressant treatment. These factorsindicates that it is desperately needed to improving our knowledge of thepathophysiology of MDD, which will aid in a more accurate earlierdiagnosis and more appropriate treatment regimens for this mental disorder.As the representative technologies in system biology research, proteomicsand metabolomics are considered to be powerful tools in exploring thedisease pathophysiology and discovering the potentially diagnosisbiomarkers. PurposeThe present study aims to screening the depression associated plasmaproteins and amino acids based on comparative proteomic and metabolimcanalysis, and further investigating the possible roles of these proteins andamino acids in the pathophysiology of MDD and their probable diagnosticpotentiality for MDD.MethodsThis experiment was divided into three parts: firstly, comparativeproteomic analysis of plasma from major depressive patients based ontwo-dimensional electrophoresis; secondly, comparative proteomic analysisof plasma from major depressive patients based on iTRAQ; thirdly,Determination of plasma free amino acids of major depressive patients basedon iTRAQ.1. Subject enrollmentAccording the criteria of subject inclusion and exclusion, majordepressive patients and healthy controls were recruited from the Departmentof Psychiatry and the Medical Examination Center of the First AffiliatedHospital of Chongqing Medical University, respectively. For proteomicexperiment, a total of42subjects (21per group) were enrolled. And51ofsubjects (depressed patients:26, healthy controls:25) were recruited forplasma amino acid determination.2. Blood sample collection and storageFasting blood samples (approximately4mL) were collected into6mL EDTA tubes between08:00and10.00and separated for plasma within1hour of collection. The centrifuge parameters are1500g*15min. Thealiquoted plasma samples were stored at-800C until analysis.3. Comparative proteomic analysis of plasma from major depressivepatients based on two-dimensional electrophoresis.Equal volumes of plasma from the two groups were pooled respectively.The pooled samples were purified using a commercial kit bought fromBio-Rad. Then equal proteins (500μg) were subjected to two-dimensionalelectrophoresis analysis, and further analyzed with PDQuest software. Thedifferentially expressed proteins were digested and identified with massspectrometry.4. Comparative proteomic analysis of plasma from major depressivepatients based on iTRAQ.Equal volumes of plasma from patients group and control group werepooled, respectively. Samples from the two groups were immunedepleted ofseven high abundance proteins (albumin, IgG, antitrypsin, IgA, transferrin,haptoglobin and fbrinogen), labeled with iTRAQ tags and then analyzed bymulti-dimensional liquid chromatography-tandem mass spectrometry. Theproteomic results were further analyzed with MetaCore database andvalidated by immunoblotting or enzyme-linked immunoadsorbent assays.5. Plasma free amino acid determination based on iTRAQ.The plasma samples were precipitated, and then the supernatants were labeled with the iTRAQ tags. The mixture of labeled samples andpre-labeled standard amimo acids were subjected to liquid chromatographicseparation and identified by tandem mass spectrometry. The data wereanalyzed by MarkerViewTM software (version1.4.2) and SPSS16.0statistics package.Result1. Based on the PDQuest analysis of the two-dimensional gelelectrophoresis maps, the protein spots found in the gels from patients groupwere189,197and185, respectively. And for control group,208,204and205of protein spots were found, respectively. The matching rate between thetwo groups was81%. However, no dysregulated proteins were discoveredbetween the two groups based on this approach.2. A amount of nine proteins, namely apolipoprotein D, afamin,apolipoprotein B100, α-1B-glycoprotein, isoform1of vitamin D-bindingprotein, ceruloplasmin, histidine-rich glycoprotein, semaphorin-3F,andα-2-macroglobulin, were found to be differentially expressed betweenthe two groups. The latter four of the nine proteins were down-regulated inthe depressed patients.3. The principal functional roles of the dysregulated proteins weremainly associated with lipid metabolism and immunoregulation. Thevalidation results showed that the statistical results of the four proteins,namely afamin, apolipoprotein D, vitamin D-binding protein and ceruloplasmin, were consistent with the proteomic results when analyzed inpooled samples used for iTRAQ analysis. These four proteins were furtheranalyzed in all42individual samples, the expression trend of the fourproteins were consistent with the iTRAQ results. However, they did notreach statistic significance. Additionally, the ELISA results ofapolipoprotein B100and α-2-macroglobulin were consistent with theproteomic findings.4. Compared to control group, plasma levels of three amino acids(tryptophan, lysine and γ-aminobutyric acid) were significantly decreased indepressed patients. Significant inverse correlations were foundbetween the depression severity assessed by HDRS scores andlevels of β-aminoisobutyric acid and aspartate in depressedpatients. A logistic regression model comprised of tryptophan, glutamineand cysteine, discriminated patients from controls with an areaunder the receiver-operating-characteristic curve of0.900.Conclusion1. The presentation of low abundance proteins in plasma was severelyinterfered by the high abundance proteins, especially albumin and IgG.Depletion of high abundance proteins is a basic step for plasma comparativeproteomics.2. Comparative proteomics is a promising strategy for investigating thepathophysiological process of major depression and screening the depression biomarker(s). Early perturbation of lipid metabolism andimmunoregulation may be involved in the pathophysiology of majordepression. Two proteins, namely apolipoprotein B100andα-2-macroglobulin may be potential biomarkers of major depressivedisorder.3. The perturbation of tryptophan, lysine and γ-aminobutyric acid maybe involved in the onset of major depressive disorder. The plasma level ofβ-aminoisobutyric acid and aspartate may be used to evaluate the severity ofmajor depression. Plasma amino acid profiling has the potentiality ofdetection of major depression.4. System biological approaches, such as proteomics and metabolomics,are powerful tools for exploring disease pathophysiology and screeningdisease biomarkers.