Proteomic approach in the search of new cardiovascular biomarkers

With the increasing incidence of cardiovascular diseases worldwide, specifically atherosclerosis and heart failure, the search for novel biomarkers remains a priority. As opposed to complex diagnostic techniques that may not be suitable to be applied to the wider population, biomarkers are useful for population screening. The search for novel biomarkers is based on knowledge of the molecular and cellular processes that take place in the development of a specific disease. Atherosclerosis and heart failure are characterized by a long period of silent disease progression, allowing early diagnosis and the potential of early therapeutic intervention. The use of the so-called proteomic techniques allows not only protein identification but partial characterization, which includes expression and also post-translational modification of these proteins. This allows for the discovery of previously unknown proteins involved in cardiovascular diseases, including some that may be suitable to be used as biomarkers. However, to approach this issue, we have to overcome difficulties such as tissue heterogeneity (vessel wall or myocardium) and the lack of fresh human samples. We discuss the proteomic study of human plaques, secreted proteins by pathologic and normal vessel wall, and left ventricular hypertrophy as potential sources of new biologic markers of cardiovascular disease.     

Proteomic analysis of gliomas

Primary malignant brain tumours (anaplastic glioma and glioblastoma) display heterogenous histopathology and diverse genetic abnormalities. These tumours remain incurable with no significant improvement in median survival times in the last 20 years, despite significant technological advances in surgery and radiotherapy, and mechanistic insights into their aetiology. Recent clinical trials suggest molecular characterization of tumours is essential in guiding both therapy and predicting prognosis. Genetic insight into tumour biology and increasingly proteomic technology has opened new avenues for novel applied clinical research. Protein expression in human malignant glioma and matched normal brain tissues can now be reliably analysed using quantitative proteomic techniques, the most accessible of which is two-dimensional gel electrophoresis (2DGE) and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry from which differentially expressed proteins can be identified and characterized. The potential of using differential proteomic profiling in gliomas to identify prognostic markers and to gain insight into tumour biology is currently being investigated. The current status of proteomic technology, its application to gliomas and the utility of such translational studies is reviewed.     

Proteomic analysis in pediatric renal disease

Children with renal disease have tremendous potential for recovery, particularly when disease processes are detected early in the disease course. However, invasive diagnostic maneuvers can be challenging, especially in younger children who may require general anesthesia. Urine proteomics technologies present an opportunity to discover noninvasive yet informative diagnostic and prognostic markers of renal disease in children. In this article we review current concepts regarding the normal urine proteome, followed by an overview of urine proteomics as applied to nephrotic syndrome, and conclude with a discussion of some of the challenges of performing proteomic profiling on nephrotic urine, with its inherent abundance of proteins.     

结直肠癌转移生物标记物的蛋白质组学研究

目的应用蛋白质组学技术寻找结直肠癌转移潜在标志物,研究氟尿嘧啶（5-Fu）对差异蛋白表达的影响,并初步探讨结直肠癌的转移机制。方法常规培养Lovo及SW480细胞至指数生长期后提取蛋白。采用MTF法检测5-Fu对此两种细胞的半数抑制浓度（IC50）,分别用IC50的5-FU干预Lovo和SW480细胞后提取蛋白。对所提取的蛋白进一步行二维凝胶电泳,选取表达有显著差异的点进行质谱检验和生物信息学分析。采用Western blot及免疫荧光验证5-FU作用前后两种细胞显著差异蛋白的表达。结果二维凝胶电泳和质谱分析成功鉴定出11种差异蛋白．根据预设条件筛选出：核内不均匀核糖核蛋白K（hnRNP K）、蛋白质二硫键异构酶（PDI）。Western blot及免疫荧光实验显示;Lovo细胞株中hnRNPK蛋白表达较SW480高,PDI蛋白表达较SW480低：5-Fu干预后．Lovo细胞株中hnRNP K蛋白表达下调程度较SW480显著．免疫荧光强度减弱,SW480细胞株中PDI蛋白表达上调幅度较Lovo细胞显著,免疫荧光强度增强。结论hnRNP K和PDI表达在不同转移潜能结直肠癌细胞系Lovo和SW480存在显著差异,5-Fu干预后其表达有着规律性改变。     

瘢痕疙瘩与正常皮肤的蛋白质组学研究

目的 通过比较瘢痕疙瘩与正常皮肤组织的蛋白质组表达差异,筛选出与瘢痕疙瘩产生相关的蛋白质.方法 2010年1月至6月运用蛋白质组学技术,对8例瘢痕疙瘩组织和3例正常皮肤组织进行差异双向凝胶电泳(2D-DIGE),选择差异蛋白质斑点,进行基质辅助激光解离飞行时间(MALDI-TOF/TOF)质谱分析和生物学信息分析.结果 成功建立瘢痕疙瘩和正常皮肤组织的双向凝胶电泳图谱,瘢痕疙瘩和正常皮肤组织凝胶电泳图谱中平均蛋白质斑点数分别为2978和3053,其中表达差异超过4倍的斑点共有40个,质谱分析和数据库检索共鉴定出32种蛋白质,包括上调蛋白有20种,下调蛋白有12种.从功能上可分为载体蛋白(3种)、信号转导蛋白(4种)、增殖凋亡相关蛋白(2种)、细胞骨架蛋白(6种)、细胞外基质蛋白(8种)、免疫因子(3种)、肿瘤相关蛋白(2种)和未知功能蛋白(4种).结论 蛋白质组学能较好地显示瘢痕疙瘩与正常皮肤组织间的蛋白质表达差异.对这些差异蛋白质进一步深入研究,将有助于揭示瘢痕疙瘩的发病机制,也为发现新的治疗靶点提供线索和依据.

Abstract：

Objective To investigate and search correlative proteins of keloid by comparing the results of differential proteomic analysis between keloid and normal skin. Methods From January 2010 to June 2010 two-dimensional gel electrophoresis was used to define patterns of protein expression in keloid skin from 8 patients and matched normal skin from 3 patients. Differential expression protein spots were showed and analyzed by matrix-assisted laser desorption ionization-time of flying/time of flying(MALDI-TOF/TOF) mass spectrometry. Results This study succeeded to provide a two-dimensional protein profiling comparison between normal skin and keloid. Gel-analysis software identified an average of 2978 spots in keloid while 30S3 spots in normal skin and statistical filtering yielded 40 spots of a 4-fold change, 32 of which were identified by using mass spectrometry, 20 were up-regulated and 12 were down-regulated. Functional analysis revealed that these proteins could be fractionated to carrier proteins (3 proteins), signal transduction proteins (4 proteins) , proliferation and apoptosis related proteins (2 proteins) , cytoskeleton proteins(6 proteins) , extracellular matrix proteins(8 proteins) , immunity related proteins (3 proteins) , tumor related proteins (2 proteins) , and function unknown protein (4 proteins). Conclusions Proteomic analysis can identify the proteins with variance of keloid versus normal skin. The further research to these differential proteins may help reveal the pathogenesis of keloid and provide new treatments for keloid.     

Proteomic analysis of the abdominal aortic aneurysm wall

Purposes

A ruptured AAA (rAAA) is a common cause of death in males over 60 years of age, and the global mortality from rAAA exceeds 80 %. The pathological processes occurring in the wall of the developing AAA are still unclear. The potential pathophysiological mechanisms underlying aortic aneurysms have been examined by many studies using immunohistochemistry and were, therefore, targeted at specific, preselected protein antigens.

Methods

We collected samples of tissue from anterior wall of an aneurysm sac from 15 patients indicated for AAA resection (group A) during the period from 2010 to 2011. These samples were subjected to a proteomic analysis. In addition, we collected control samples of identical aortic tissue from 10 heart-beating deceased organ donors (group B).

Results

A total of 417 differentially expressed protein fractions were identified, 18 of which were only detected in the healthy controls, while 85 were specific for aneurysm tissue and 314 were detectable in both groups. In 175 protein fractions, the gel-derived spot volumes differed significantly between aneurismal and healthy aortic tissue.

Conclusions

We found a significant difference in the proteome of the AAA tissue and non-dilated aortic tissue. We demonstrated that the AAA proteome is considerably richer and more varied than the healthy and atherosclerotic aorta. We believe that our results clearly demonstrate a completely different etiopathogenesis of atherosclerosis and aneurismal disease.     

Proteomic analysis to identify biomarker proteins in pancreatic ductal adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fifth most common cause of death from cancer in Korea. PDAC is difficult to diagnose at an early stage and even more difficult to cure. Thus, there is an urgent need to identify molecular targets for early diagnosis and effective treatment. The objectives of this study were to identify differentially expressed biomarker proteins of PDAC using proteomic analysis, to validate the identified biomarker proteins associated with carcinogenesis using western blot analysis and to evaluate clinical factors influencing expression of candidate biomarker proteins. Methods: In the present study, we carried out proteomic analysis in 10 pairs of PDAC specimens with matching adjacent normal tissues to clarify the different patterns of protein expression. The proteins were separated by high‐resolution 2‐D polyacrylamide gel electrophoresis (2D PAGE) and the differentially expressed proteins were identified by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Differential expression of candidate biomarker proteins associated with carcinogenesis was further validated using western blot analysis. Standard statistical analysis was carried out in an attempt to establish a correlation between clinical variables and expression of candidate biomarker proteins. Results: Analysis of PDAC and the adjacent normal tissues showed reproducibly similar proteomic patterns for each group. Approximately 700 spots each were seen by silver‐stained gels from both PDAC and normal tissues. Differentially expressed protein spots were gel digested and identified by MALDI‐TOF MS. Twenty‐five proteins were identified, of which five proteins (galectin‐1, enolase‐2, α‐1‐antitrypsin, N‐myc interactor, peroxiredoxin‐4) were previously reported as being differentially expressed either at the mRNA level or protein level in human cancer. The five proteins were selected for candidate biomarker proteins related to carcinogenesis. These proteins were further validated by western blot analysis. Among the candidate biomarker proteins, galectin‐1 expression was highly correlated to histology (P = 0.019), T stage (P = 0.047), N stage (P = 0.033) and American Joint Committee on Cancer stage (P = 0.011). Conclusion: Differentially expressed 25 proteins in PDAC were identified using proteomic analysis and five proteins related to carcinogenesis were validated by western blot analysis. galectin‐1 expression was highly correlated to tumour histology and stage.     

心血管外科的临床研究与决策

心血管外科是一种高风险的学科,在临床工作中可能会遇到各种各样的患者和病情,处理不当,可能会给患者带来很大的危害甚至危及生命.因此,做好临床研究工作,做出正确的决策,无论对于学科发展建设,还是对于提高手术疗效都很重要.     

Proteomic analysis of peritoneal dialysate fluid in patients with dialysis-related peritonitis

The prognosis of uremia patients on continuous ambulatory peritoneal dialysis (CAPD) is related to frequent peritonitis rate. Frequent peritonitis will lead to peritoneum failure, making CAPD unfeasible. We have performed proteomic profiling of peritoneal dialysis effluent samples from a cross-section of CAPD patients with and without peritonitis in order to identify biomarkers of peritonitis. We performed 2D gel electrophoresis and surface-enhanced laser esorption/ionization time of flight mass spectrometry (SELDI-TOF MS) on peritoneal dialysis effluent from 16 subjects with peritonitis. A genetic algorithm search of principal component space revealed a group of a peak distinguishing peritonitis-positive subjects, with mass/charge (m/z) values of 11,117.4. Our analyses identified the peak at m/z 11,117.4 with an accuracy of 95% for classifying peritonitis. Mass spectrometric analysis of peritonitis PDE samples identified the 11,117.4 protein as beta2-microglobulin (B2M). Using an unbiased protein profiling approach, we have validated previously reported findings of B2M as a biomarker associated with CAPD peritonitis. Prospective studies are warranted to establish additional biomarkers that would be predictive of peritoneal dialysis peritonitis. Besides, extending the study to a larger number of patients with subgroup analyses may yield additional information of the peritoneal dialysate proteins in association with dialysis adequacy, residual renal function, nutritional status, and risk of peritoneal infection.     

Proteomic analysis of seminal plasma in men with different spermatogenic impairment

Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system including male infertility. The identification and characterisation of differentially expressed proteins in seminal plasma of man with normal and impaired spermatogenesis can help in the elucidation of the molecular basis of male infertility. We compared the protein expression profiles of seminal plasma from four different groups of men as follows: normozoospermic, asthenozoospermic, oligozoospermic and azoospermic groups, using two-dimensional differential gel electrophoresis (2-D DIGE). We found eight proteins with statistically significant increased expression in azoospermia compared with at least one of the other studied groups. The differentially expressed spots were fibronectin, prostatic acid phosphatase (PAP), proteasome subunit alpha type-3, beta-2-microglobulin, galectin-3-binding protein, prolactin-inducible protein and cytosolic nonspecific dipeptidase. Notably, PAP was increased in patients with azoospermia compared with that of all other groups. We have observed no statistically significant differences in protein expression between three of the groups: normozoospermic, oligozoospermic and asthenozoospermic. We suggest that the identified panel of proteins in our study especially PAP have a strong potential to be used as azoospermia markers. However, further investigations will be necessary to validate these markers in samples of larger and independent patient cohorts and to clarify their role in the pathogenesis of male infertility.     

Proteomic analysis of urine in kidney transplant patients with BK virus nephropathy

The differentiation of BK virus-associated renal allograft nephropathy (BKVAN) from acute allograft rejection (AR) in renal transplant recipients is an important clinical problem because the treatment can be diametrically opposite for the two conditions. The aim of this discovery-phase biomarker development study was to examine feasibility of developing a noninvasive method to differentiate BKVAN from AR. Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry analysis was used to compare proteomic profiles of urine samples of 21 patients with BKVAN, 28 patients with AR (Banff Ia to IIb), and 29 patients with stable graft function. SELDI analysis showed proteomic profiles that were significantly different in the BKVAN group versus the AR and stable transplant groups. Peaks that corresponded to m/z values of 5.872, 11.311, 11.929, 12.727, and 13.349 kD were significantly higher in patients with BKVAN. Bioinformatics analyses allowed distinction of profiles of patients with BKVAN from patients with AR and stable patients. SELDI profiles also showed a high degree of reproducibility. Proteomic analysis of urine may offer a noninvasive way to differentiate BKVAN from AR in clinical practice. The identification of individual proteomic peaks can improve further the clinical utility of this screening method.