Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium

Purpose: To exploit the potential of proteomics to identify and study additional yet‐unidentified important proteins present in human endometrium. Experimental design: The proteome of human endometrium would be established using 2‐DE and MALDI and the data analyzed to identify differential protein expression in the proliferative and secretory phase of the menstrual cycle using PDQuest software and MALDI. Results: In the present work, 2‐DE of human endometrium protein led to the resolution of over 200 spots. Subsequent MALDI analysis of 215 spots allowed the identification of 194 proteins. A total of 57 out of the 215 spots were found to be differentially expressed, out of which 49 could be identified using MALDI. These differentially expressed proteins included structural proteins, molecular chaperones, signaling proteins, metabolic proteins, proteins related to immunity, RNA biogenesis, protein biosynthesis and others. The differential expressions of seven representative proteins in secretory and proliferative phase endometrium tissue were confirmed by immunoblot analysis. Conclusion and clinical relevance: This study establishes the 2‐D proteome of human endometrium represented by 194 identified protein spots. The present data provides an important clue towards determining the function of these proteins with respect to endometrium related diseases.     

Alpha-1-antitrypsin and complement component C7 are involved in asthma exacerbation

Asthma is the most common chronic disorder in childhood and asthma exacerbation is an important cause of childhood morbidity and hospitalization. Allergic responses are known to be biased toward T-helper type 2 in asthmatics; however, the pathogenesis of asthma is not simple, and our understanding of the disease mechanism remains incomplete. The aim of the present study was to identify protein expression signatures that reflect acute exacerbation of asthma. Plasma was taken twice from pediatric asthmatic patients, once during asthma exacerbation and once during a stable period. Plasma was also taken from healthy children as a control. The protein profiles of plasma during asthma exacerbation were analyzed by 2-DE and 49 spots were differentially expressed during asthma exacerbation. Thirty-eight of the spots were successfully identified by MALDI-TOF MS. Proteins up- or down-regulated during asthma exacerbation were involved in responses to stress and pathogens, in the complement and coagulation cascades, and in acute-phase responses. Among the differentially expressed proteins, up-regulation of alpha-1-antitrypsin and complement component C7 was confirmed by nephelometry and ELISA. Our present results suggest that protease inhibitors and complement components may be involved in asthma exacerbation, and plasma level of alpha-1-antitrypsin may be a potential biomarker for asthma.     

Identification of differentially expressed proteins in papillary thyroid carcinomas with V600E mutation of BRAF

BRAF, a serine/threonine kinase of the RAF family, is a downstream transducer of the RAS-regulated MAPK pathway. V600E mutation of BRAF protein is the most common genetic alteration occurring in papillary thyroid carcinomas and is prognostic of poor clinicopathological outcomes. Protein expression in the subclass of PTC bearing the BRAF(V600E) mutation was investigated by using 2-DE and MS/MS techniques and compared to that of matched normal thyroid tissues from seven patients. 2-D gel image analysis revealed that the expression of eight polypeptide spots, corresponding to five proteins, were significantly underexpressed in PTC bearing BRAF(V600E) mutation whereas 25 polypeptides, representing 19 distinct proteins, were significantly upregulated in tumour tissue, as compared to normal thyroid. Among the differentially expressed polypeptides, mitochondrial proteins, ROS-scavenger enzymes, apoptosis-related proteins as well as proteins involved in tumour cell proliferation were identified. Although dissimilarities between the present results and those previously reported can be ascribed to the use of different 2-DE techniques, the possibility that BRAF(V600E) mutation is responsible for changes in protein expression distinct from those induced by other oncogenes cannot be ruled out.     

Quantitative analysis of myofilament protein phosphorylation in small cardiac biopsies

Phosphorylation of cardiac myofilament proteins represents one of the main post‐translational mechanisms that regulate cardiac pump function. Human studies are often limited by the amount of available tissue as biopsies taken during cardiac catheterization weigh only 1 mg (dry weight). Similarly, investigation of time‐ (or dose‐) dependent changes in protein phosphorylation in animal studies is often hampered by tissue availability. The present study describes quantitative analysis of phosphorylation status of multiple myofilament proteins by 2‐DE and Pro‐Q® Diamond stained gradient gels using minor amounts (?0.5 mg dry weight) of human and pig cardiac tissue.     

Proteomic profiling of cancer of the gingivo-buccal complex: Identification of new differentially expressed markers

Tobacco-related oral cancer is the most common cancer among Indian males, gingivo-buccal complex (GBC) being the most affected subsite due to the habit of chewing tobacco. Proteins from the lysates of microdissected normal and transformed epithelium from clinically well-characterized tissue samples of the GBC were separated by two-dimensional gel electrophoresis to identify differentially expressed proteins. Eleven protein spots showed differential expression, which could withstand the stringency of statistical evaluation. The observations were confirmed with additional tissues. Nine of these differentiators were identified by MS as lactate dehydrogenase B, α-enolase, prohibitin, cathepsin D, apolipoprotein A-I, tumor protein translationally controlled-1, an SFN family protein, 14-3-3σ and tropomyosin. Cluster analysis indicated that these proteins, as a coexpressed set, could distinguish normal and transformed epithelium. Functionally, these differentiator molecules are relevant to the pathways and processes that have been previously implicated in oral carcinogenesis and could therefore be investigated further as a panel of markers for management of cancer of the GBC.     

Proteomic analysis of sera of asymptomatic,early‐stage patients with Wilson's disease

Wilson's disease (WD) is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues, leading to significant oxidative stress and tissue damage. To date, several diagnostic biomarkers for WD such as serum ceruloplasmin, serum or urine copper levels and copper content in liver have been identified. However, these biomarkers may not be convincing for the diagnosis in some WD patients. To identify additional novel diagnostic biomarkers, we compared the serum protein profiles of asymptomatic childhood WD patients (n=20), without neurologic manifestation or liver cirrhosis, with normal controls (n=13). Fourteen spots, five up‐regulated and nine down‐regulated (>2‐fold), were differentially expressed in WD patients in comparison to normal control on 2‐DE. Among them, three spots were down‐regulated in both male and female WD. MS/MS analysis revealed that the three spots were complement component C3, complement factor B and alpha‐2 macroglobulin. By comparative proteome analysis, complement component C3, complement factor B and alpha‐2 macroglobulin, which are related to oxidative stress and inflammation, turned out to be good candidates for novel diagnostic biomarkers for early stages of WD.     

Identification of distinctive protein expression patterns in colorectal adenoma

Purpose: As a pre‐malignant precursor, adenoma provides an ideal tissue for proteome profiling to investigate early colorectal cancer development and provide possible targets for preventive interventions. The aim of this study was to identify patterns of differential protein expression that distinguish colorectal adenoma from normal tissue. Experimental design: Twenty paired samples of adenoma and normal mucosa were analysed by 2‐DE and MALDI‐TOF/TOF MS to detect proteins with ≥2‐fold differential expression. Results: Four proteins were up‐regulated in adenoma (Annexin A3, S100A11, S100P and eIF5A‐1) and three were down‐regulated (Galectin‐1, S100A9 and FABPL). S100P, galectin‐1, S100A9 and FABPL expression was localised by immunohistochemistry. Conclusions and clinical relevance: Distinctive patterns of in vivo protein expression in colorectal adenoma were identified for the first time. These proteins have important functions in cell differentiation, proliferation and metabolism, and may play a crucial role in early colorectal carcinogenesis. The ability to recognise premalignant lesions may have important applications in cancer prevention.     

Proteome analysis of human foetal, aged and advanced nuclear cataract lenses

The most complete proteome of human lenses has been compiled using 2-D LC-MS/MS analysis of foetal, aged normal and advanced nuclear cataract lenses. A total of 231 proteins were identified across all lens groups, including 112 proteins that have not been reported previously. Proteins were grouped according to their PANTHER molecular function classification in order to facilitate comparisons. Previously unreported N-terminal acetylation was detected in a number of proteins, with the majority being associated with the prior removal of a methionine residue. This pattern of proteolysis may indicate that methionine aminopeptidase activity is present in human lenses. Acetylation is likely to aid in the stability of proteins that are present in the lens for many decades. Protein sequences were also used to interrogate the three human lens cDNA libraries publicly available. Surprisingly, 84 proteins we identified were not present in the cDNA libraries.     

Highlights of the first BSPR London Regional Meeting and Fifth Imperial Proteomics Day

Now that the genomics revolution is tailing off proteomics promises an even more radical transformation of biological and medical research. Ultimately, the clues to the mysteries of biological processes will lye within the proteins present at a given time. Proteomics, the characterization of complex protein mixtures, builds on a wide range of expertise from protein chemistry to mass spectrometry and bioinformatics. Each step from sample preparation, high resolution protein separation to protein identification is underpinned by the latest technological developments in protein separation and mass spectrometry and supported by powerful computational image and data analysis algorithms which requires large scale data management and storage facilities. The success of proteomics depends on further developments in technology which need a wide range of scientific expertise. This meeting, the first BSPR London Regional Meeting, brought together scientists from a broad background who share a common interest in novel solutions for the characterization of complex protein mixtures.     

Identification of new epidemiological molecular markers by comparative proteomics of serogroup A meningococcal isolates from three pandemic waves

We previously described the first reference map for the proteome of one strain of serogroup A Neisseria meningitidis (MenA), a major cause of epidemic meningitis in humans. As a preliminary finding, in that work we noted that 2‐DE protein maps of closely related MenA isolates from different epidemics spreads could be easily compared to detect minor differences and that 2‐DE phenotypes attributable to the well‐known epidemiological marker tbpB agreed with the genoclouds model of MenA epidemiological variation during pandemic waves. We explored here the possibility that an extended comparative study of 2‐DE maps of isolates representative of the nine genoclouds described by Achtman and collaborators could be used to discriminate between strains otherwise undistinguishable. We showed the example of 14 proteins with different 2‐DE spot patterns in different genoclouds that could be considered as putative tracers for alike‐strains discrimination. We introduce the novel concept that comparative proteomics can be useful in identifying new epidemiological markers for N. meningitidis.     

Protein profiling of low-density lipoprotein from obese subjects

Although obesity and high levels of low-density lipoprotein (LDL) are well-known risk factors for cardiovascular disease, the precise role(s) of different LDL constituents in obesity has not been explored. In the present study, we compared the LDL proteome of healthy control adults (body mass index<25) and obese subjects (body mass index>30). LDL was isolated by density-gradient ultracentrifugation and proteins were separated with 2-D PAGE, quantified, and identified by peptide mass fingerprinting using MALDI-TOF MS. A new LDL-associated protein was identified as transthyretin and found to be significantly more abundant in LDL from the obese subjects. In addition, LDL from the obese subjects contained relatively more α(1) -antitrypsin, apo J, apo C-II, than LDL from controls, and also more of an acidic isoform (pI/Mr; 5.2/23 100) of apo A-I. On the other hand, the relative amounts of apo A-IV and the major isoform of apo A-I (pI/Mr; 5.3/23 100) were significantly less in LDL from the obese subjects. Apo E was less and non-sialylated apo C-III more abundant in LDL from obese men than control men, while there were no such differences between LDL from obese and control women. These findings illustrate that obesity is not only associated with increased LDL-cholesterol levels but also with alterations in the LDL protein composition. The presence of transthyretin in LDL from obese subjects may reflect over-nutrition and affect the lipid metabolism in obesity.     

Proteomics analysis of plasma for potential biomarkers in the diagnosis of Alzheimer's disease

The objective of this study was to search for biological markers associated with Alzheimer's disease (AD). Plasma specimens obtained from ten pathologically diagnosed AD patients and ten non-demented (ND) control subjects were analyzed by a combination of 2-DE and MS. This strategy allowed us to identify six plasma proteins (alpha-1-antitrypsin, vitamin D-binding protein, inter-alpha-trypsin inhibitor family heavy chain-related protein, apolipoprotein J precursor, cAMP-dependent protein kinase catalytic subunit alpha 1, and an orf) whose 2-DE spot densities were different between the AD and ND groups. Due to their involvements in AD amyloid plaque formation, the plasma concentrations of alpha-1-antitrypsin and apolipoprotein J were further validated using either ELISA or Western blot. The results revealed that the plasma levels of alpha-1-antitrypsin in AD were higher than those of controls, confirming the 2-DE findings. However, no difference in total apolipoprotein J concentration was observed between the AD and ND groups. Considering the difference in resolving power to differentially quantitate protein isoforms provided by 2-DE and Western blot, 2-DE analysis combined with MS protein identification offers distinctive advantages when a disease-related protein isoform-specific variance is investigated.     

Towards a comprehensive proteome of normal and malignant human colon tissue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.     

The discovery of biomarkers for type 2 diabetic nephropathy by serum proteome analysis

Diabetic nephropathy (DN) is a serious kidney complication of diabetes, and constitutes the leading cause of end-stage renal disease. The earliest clinical evidence of DN is microalbuminuria, a term which refers to the appearance of small but abnormal amounts of albumin in the urine. However, screening methods for DN, such as biomarker assays, are yet to be developed for type 2 DN. In the present study, in an attempt to identify the biomarkers for initial diagnoses of type 2 DN, the protein profiles of human sera collected from 30 microalbuminuric type 2 diabetic patients were compared with those collected from 30 normoalbuminuric type 2 diabetic patients, via 2-DE. As a result, a total of 18 spots were determined to have different protein levels in the microalbuminuric patients. Twelve spots had lower protein levels of approximately 50%, and the other six had higher levels of approximately 100-300% as compared to the spots of normoalbuminuric patients. These spots were identified with ESI-Q-TOF (ESI-quadrupole-TOF) MS. Among the identified proteins, vitamin D-binding protein (DBP) and pigment epithelium-derived factor (PEDF) were verified by Western blotting. The results of this study indicate that the DBP may be employed as diagnostic and monitoring biomarkers of type 2 DN, contingent on further study into the matter.     

Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end-stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2-DE and identified with ESI-Q-TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic acid phosphatase precursor, the ribonuclease and the kallikrein-3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig κ chain C region, Ig κ chain V-II region Cum, Ig κ-chain V-III region SIE, carbonic anhydrase 1, plasma retinol-binding protein, β-2-microglobulin precursor, β-2-glycoprotein 1. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage.     

State of the art of 2D DIGE

Difference gel electrophoresis enables the accurate quantification of changes in the proteome including combinations of PTMs and protein isoform expression. Here, we review recent advances in study design, image acquisition, and statistical analysis. We also compare DIGE to established and emerging mass spectrometric analysis technologies. Despite these recent advances in MS and the still unsolved limitations of 2DE to map hydrophobic, high molecular weight proteins with extreme pIs, DIGE remains the most comprehensive top-down method to study changes in abundance of intact proteins.     

Identification of nasopharyngeal carcinoma antigens that induce humoral immune response by proteomic analysis

A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.     

基于FPGA的乒乓游戏机设计

本文使用FPGA芯片来模拟实际的乒乓球游戏。本设计是基于Altera公司的FPGA CycloneⅡ芯片EP2C35的基础上实现,运用Verilog HDL语言编程,在QuartusⅡ软件上进行编译、仿真,最终在Altera公司的DE2开发板上成功实现下载和调试。     

Proteomic analysis of pericardial effusion: Characteristics of tuberculosis-related proteins

The aim of this study has been designed to identify the tuberculosis (TB)-related proteins in pericardial effusion by proteomic approaches. TB is one of the major infectious diseases causing pericardial effusion. This study details protein profiles in pericardial effusion from three TB patients and three heart failure patients. Pericardial effusions were analyzed using 2-DE combined with the nano-HPLC-ESI-MS/MS. Eleven protein spots with differential expression in pericardial effusion were identified between the two groups of TB and heart failure patients (the control group). Seven protein spots were upregulated and four were downregulated. The composition of the pericardial effusion proteome may reflect the pathophysiological conditions affecting the progression of tuberculous pericarditis. The proteins in the tuberculous pericardial effusion with differential expression may serve as new and direct indicators of drug treatment. A possible conclusion is indicated that fibrinogen may play an important role for fibrin assembly in tuberculous pericardial effusion.