Differential Proteome Analysis Of Human Serum From Diabetic Retinopathy

BackgroundDiabetic retinopathy(DR) is one of the most common and severe microvascular complications of diabetes mellitus. It mainly manifests with decreased sight, progressive retina impairment and permanent loss of sight. Its manifestations could be microaneurysms, hemorrhage, exudation and edema on retina. Eventually, retinal neovessels, folds, and detachment afterwards lead to vision loss. DR is the important reason leaing to vision loss among diabetes mellitus patients. When diagnosed diabetes mellitus 10 to 15 years, almost all type 1 diabetes mellitus(T1DM) patients and more than 60% type 2 diabetes mellitus(T2DM) patients may suffer from DR. DR seriously affects the patients’ life, increase medical expenses and social burden. At present, the ideal method treating DR is still not found. In clinical practice, we noticed that lots of patients with DR didn’t show obvious symptoms in early stage. Therefore, it is with great significance to unfold DR pathogenesis, and verify the protein biomarkers on its early diagnosis as well as prognosis. The pathogenesis of DR is very complex. For many year, domestic and foreign researchers have done a large number of animal experiments and clinical research and made some progress in this area. These former studies were convinced that the occurrence and development of DR is related to a variety of pathologenic factors like sorbitol or aldose reductase pathway, non-enzymatic glycosylation products, growth factors and hemodynamic abnormalities in retina. However, these outcomes can not explain DR pathogenesis in a further molecular level.Many studies indicate that DR is a complicated biological process including multiple factors and steps. Single molecular research can not fully clarify its mechanism. In recent years, fast development of proteomic technology has provided a reliable technological stage for high-through molecular marker research. So we are enabled to realize the studies on exploration of DR pathogenesis by multiple level and factors, seeking for DR specific molecular markers for early diagnosis, as an emerging discipline, Proteomics mainly focus on the overall level to research functions of the object. Using proteomics methods can fully reveal protein expression in tissue and cell. Currently, many researchers have established the protein profiles of diseases successfully using proteomic methods and have found some disease-related molecular markers. Constantly improved research technology, together with bioinformatics databases and analysis software provide feasible technical support for large-scale studying proteome. As far as DR is concerned, compared with the expressional differences of proteins between normal and abnormal state, a variety of factors and regulatory networks can be revealed. This systematic study has its unique advantages to research pathological mechanisms for DR.Today, most proteomic analysis on DR patients are based on vitreous humor or eye tissue from animal model. However, neither is flawless. Vitreous humor is hard to draw, and blood-retinal barrier impairment may cause biased result. Meanwhile, animal models cannot authetically duplicate the characteristics of DR patients. One the other hand, as the development of serum proteomic technology, proteomic research based on serum samples can convey the protein expression more clearly and authentically. These serum molecular markers, mainly low-abundance proteins, are closely related to the disease and promising in diagnosis, accounting for less than 1% in serum total protein. They are hard to separate and characterize for they are usually covered or interfered by albumin and immunoglobulin which account for 70% in serum total protein. Therefore, it is very important to select highly sensitive and reliable proteomic technology when doing serum proteomic analysis. The mainly supporting technologies of proteomics are two- dimensional gel electrophoresis(2-DE), mass spectrometry and bioinformatics. This kind of technology has become the most common protein separation technology. At present, there are many novel differential proteomics analysis methods, among which the difference gel electrophoresis (difference gel electrophoresis, DIGE) technology becomes one of the most popular one, because it’s not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis, but also have high reproducibility, high sensitivity, high throμghput and high dynamic range. DIGE is a method which label protein samples with different fluorescent dyes before 2-D electrophoresis, and then separate up to three different protein samples at the same time in one two-dimensional gel, The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the real biological differences, while avoid influence of systematic errors on experimental results.How to make those patients who suffer from DR getting timely and effective prevention and treatment? How to determine which patients are in need of early intervention? Is it feasible using proteomic method in study of human serum from patients with DR? As far as proteomics analysis is concerned, which important proteins are related to the pathogenesis of DR? With these problems, we chose this topic. We use 2D-DIGE separation technology, combined with matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry(MALDI-TOF-TOF) technology to screen serum specific molecular markers in T2DM patients complicated with DR. Few similar literature has been reported so far. Purpose1. To explore the feasibility using 2D-DIGE combined with MALDI-TOF-TOF in study of human serum from patients with diabetic retinopathy.2. To select differential proteins and specific molecular markers from human serum with DR through studying serum proteomics of patients with DR in various phases, so as to explore the mechanism of DR.3. To clarify the clinical significance and values of specific molecular markers from human serum through verifying differential proteins in clinical practice, so as to provide new clues when finding serum candidate markers and proteins related to the mechanism of DR.Methods1. Subjects:Twenty-four T2DM patients in accordance with inclusion and exclusion criteria were recruited. All the patients were divided into three groups:one group of 8 patients with no DR(NDR group), one group of 8 patients with non-proliferative diabetic retinopathy(NPDR group) and one group of 8 patients with proliferative diabetic retinopathy(PDR group). Additionally,8 healthy volunteer participants with matched age and sex were included in normal control group (NC group).2. Serum Sample Collection:Blood drawn from cubital vein with empty stomach was centrifuged. Upper serum was collected, charged separately and reserved at-80 ℃.3. Serum high-abundance protein removal:Serum diluted by combined buffer was incubated under room temperature. The filtered solution without albumin or IgG was collected by centrifugation. Eluent was collected by centrifugation, charged separately and reserved at-80 ℃.4. Protein sample purification:Protein sample was purified by 2-D Clean-up Kit.5. Detection of protein concentration:Protein concentration was detected by EttanTM 2-D Quant Kit. Protein contents were calculated according to standard curve and the absorbance values of protein samples.6. Protein separation by 2D-DIGE:Two DIGE gels were made, A gel and B gel. In each group, eight serum samples treated in above-mentioned way were taken and mixed by equivalent volume, then four samples were made. The four samples were mixed by equivalent volume to establish internal standard. Three kinds of special fluorescent dye, Cy2, Cy3 and Cy5 were used to conduct labeling reaction. The labeled protein samples were put in 24cm immobilized pH gradient(IPG) gel bars. Surfate-pplyacrylamide gel electrophoresis(SDS-PAGE) with gum concentration of 12.5%. Typhoon 9400 scanner was used to scan image of gel plate after electrophoresis. Cy2, Cy3 and Cy5 was scanned by laser with wavelength of 488/520nm,532/580nm and 633/670nm. The gel with most protein spots was made reference gel and matched with other gels. The matched spots were analysed in next stage of research. Analytic software DeCyder 2D 7.0 was used to do Differential In-gel Analysis(DIA). The differential protein spots were identified by protein spots difference greater than 1.5-fold in size(1.5-fold up-regulated or down-regulated). After sample volume increase, ordinary two-dimensional gel electrophoresis was done to get preparation gel. In-gel Coomssie Brilliant Blue staining was carried out after the second electrophoresis. Analytic gel was matched with preparation gel to find out matched differential protein spots. EttanTM Spot picker automatic workshop was used to dig the gels. The gel particles collected was cryo-preserved in EP tube under-20 ℃ for following mass spectrometry.7. Protein identification by MALDI-TOF-TOF MS and Database search: In-enzyme glycolysis of trypsin(Promega) was manipulated to differential protein spots. The prepared samples were done with peptide mass fingerprinting,(PMF) analysis by MALDI-TOF-TOF MS. Then ten strongest peaks were selected to obtain tandem mass spectrometry(MS/MS) data. Data of protein PMF by MS and MS/MS were searched by Mascot engine in Swiss-Prot database to get bioinformatics data.8. Verification of differential protein beta 2-glycoprotein I(β2-GPI) expression in serum by Western blot:Western blot method was applied to detect the expression level of β2-GPI in four serum samples taken randomizedly from four groups (80 samples) respectively. Specialty software Quantity One was used to determine area average optical density.in each band.9. Statistical analysis:Data are expressed as means±tandard deviation (x ± s). Multiple comparisons were analysed by one-way ANOVA. The pairwise comparison was performed with LSD test if the variance of the data was homogeneous, otherwise with Welch method. Statistical significance was set at P＜0.05. All statistical analysis was carried out by SPSS software (version 13.0). In-gel difference analysis was carried out by DeCyder v.7.0 automatically.Results1. The average protein concentration in each group:NC group:5.6mg/ml, NDR group:6.1mg/ml, NPDR group:5.8 mg/ml, PDR group:5.5mg/ml.2. Serum protein profiles established by 2D-DIGE were successfully obtained from patients with DR in various phases. The paired analysis on image by software DeCyder 2D 7.0 unveiled 1854 protein spots in gel A, and 1613 ones in gel B. Differential protein spots were sought based on the standard ratio>1.5. Twenty-six differential protein spots were detected. Compared to NC group, there were seven up-regulated and six down-regulated differential protein spots in NDR group,11 up-regulated and four down-regulated ones in NPDR group, nine up-regulated and four down-regulated ones in PDR group.3. Among the 26 differential protein spots screened by 2D-DIGE technology,24 were characterized by MALDI-TOF-TOF MS mass spectrometry and two were not. Most of them were high-abundance proteins in plasma, such as serum albumin, transferrin, immunoglobulins, fibrinogen, haptoglobin, clusterin and ceruloplasmin. Proteins with molecular weight under lOkDa were not separated. There were four proteins with low-abundance and low molecular weight, which were meaningful to the pathogenesis exploration and disease diagnosis. They were β2-GPI, alpha2-HS-glycoprotein(AHSG), alphal-acid glycoprotein(a1-AGP) and apolipoprotein A-1(apo A-1). Among them, Only β2-GPI of them manifested regularly in expression level, which was up-regulated with the development of DR. Differential protein spots expression level was evaluated on ratio>1.5(DeCyder 2D 7.0). Compared with NC group β2-GPI increased significantly in NDR group(ratio=1.54), NPDR group(ratio=2.43) and in PDR group(ratio=2.84). Compared with NDR group, β2-GPI increased significantly in NPDR group(ratio=1.58) and PDR group(ratio=1.84). Compare with NPDR group, there was no significant increase found in PDR group(ratio=1.17). β2-GPI probably takes part in the process of DR occurrence and development and it could be a candidate biomarker on DR diagnosis in early phase.4. Western blot analysis on β2-GPI:The outcomes showed that 50 kDa of β2-GPI expression was found in serum of each group:strong expression of β2-GPI level in PDR and NPDR groups, moderate expression in NDR group, and little expression in NC group. By calculated the average optical density value of NC、NDR、NPDR and PDR group, the values were 440564.9±66597.4、655561.6±85640.8、 859969.7±85461.0、 954887.4±125764.4 respectively. Compared with NC group, β2-GPI increased significantly in NDR, NPDR and PDR group(p<0.01). Compared with NDR group, β2-GPI increased significantly in NPDR and PDR group(p<0.01). Compared with NPDR group, there was no significant increase found in PDR group(p >0.05). Its change trend was consistent with the outcome of the proteomic experiment.Conclusion1. The proteomic method using 2D-DIGE combined with MALDI-TOF-TOF in study of human serum from patients with diabetic retinopathy is feasible.2. The proteomic method 2D-DIGE can not break the internal limitation based on 2-DE, which means it can hardly differentiate proteins with low molecular weight (under 10kDa)or low abundance.3. β2-GPI probably takes part in the process of DR occurrence and development and it could be a potential biomarker on DR diagnosis in early phase.Innovation point1. We successfully established the 2D-DIGE profiles of human serum protein from NDR, NPDR, PDR and healthy control groups.2. We proposed the view that β2-GPI probably tooks part in the process of DR occurrence and development, which could provide an experimental clue for further studying β2-GPI as a potential biomarker on DR diagnosis in early phase.