A combined proteomic and genetic analysis of the highly variable platelet proteome: From plasmatic proteins and SNPs

High biological variation in protein expression represents a major challenge in clinical proteomics. In a study based on 2D-DIGE, we found that the standardised abundance of only a few proteins varied by more than 50%. While some of the highest variable proteins in platelets of 52 healthy elderly were of plasmatic origin, such as albumin or haptoglobin, absence of several other high-abundant plasma proteins strongly suggests that plasma-derived proteins represent an integral part of the platelet proteome. Amongst the highly variable platelet-derived proteins, two spots were both identified as GSTO1 and assigned to either the wild-type or mutant isoform of SNP A140D. Remarkably, when the spots were considered within the respective genotype groups, their CV decreased to about the median variation. Albeit 2D-DIGE allowed correct genotyping, two individuals seemed to be GSTO1*A140 deficient. Probing 2D-Western blots with novel mAb, however, detected A140 protein as additional spot at pH 8.1, caused by the SNPs E155del and E208K. In contrast to previous studies, we show that GSTO1 protein is expressed in vivo, despite the deletion E155. Our data indicate that incorporation of exogenous proteins and genetic polymorphisms of endogenous proteins represent the main source of extreme biological variation in the platelet proteome.     

Analysis of nitroso-proteomes in normotensive and severe preeclamptic human placentas

Nitric oxide (NO) plays a key role in placental biology, and placental dysfunction is the main pathogenesis pathway for preeclampsia, yet the direct placental targets of NO actions have not been determined. Covalent adduction of an NO moiety to cysteines, termed S-nitrosylation (SNO), is emerging as a key route by which NO can directly modulate protein functions. This study was conducted to analyze global S-nitroso (SNO)-proteins in human placentas and to determine if their levels differ in normotensive versus severe preeclamptic placentas. Although total nitrite/nitrate increased, total levels of SNO-proteins and nitrosylated forms of endothelial NO synthase and heat shock protein 90 were decreased by preeclampsia. We further compared normotensive and preeclamptic placental nitroso-proteomes (total SNO-protein profiles) by using a biotin and CyDye switch test combined with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and identified SNO-proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Numerous SNO-proteins were displayed as spots on 2D-DIGE gels. One hundred spots of interest were excised; 46 spots were identified, of which 8 spots were novel SNO-proteins; levels of 15 spots were increased, and 6 spots were decreased, and the rest were unchanged by preeclampsia. Pathway analysis suggested that placental SNO-proteins are involved in regulating various cellular functions including protein synthesis, cell movement and metabolism, cell signaling, and other functions. These data therefore show for the first time that SNO is a crucial mechanism by which NO directly regulates placental proteins linked to various biological pathways. The significantly altered placental nitroso-proteome in preeclampsia suggests that SNO plays a role in the placental pathophysiology in preeclampsia.     

Application of fluorescence difference gel electrophoresis technology in searching for protein biomarkers in chick myopia

The lens-induced myopia (LIM) in response to concave lens (negative lens) is a well established animal model for studying myopia development. However, the exact visual and neurochemical signaling mechanisms involving myopic eye growth are yet to be elucidated. The feasibility of applying a novel two-dimensional fluorescence difference gel electrophoresis technique for global protein profilings and a search for differential protein expressions in LIM were explored in the present study. Two-dimensional polyacrylamide gel electrophoresis was performed employing a "minimal Lysine labeling" approach and a reverse CyeDye experimental protocol using retinal tissue from chicks. The retinal protein profiles between myopic and control eyes were found to be very similar. More than a thousand protein spots could be detected on a 2D gel. Sixteen and ten protein spots were found to be up-regulated and down-regulated respectively in the myopic eyes according to our preset criteria with the inclusion of an internal pool standard. About 65% of those filtered spots could be successfully identified by peptide mass fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry . Most of the differentially expressed proteins were found to be related to cytoskeletal or oxidative functions. According to the prediction of subcellular locations, most of them (about 84%) were classified as cytoplasmic proteins. The cellular functions for those differentially expressed proteins were reported and their possible involvements in the compensated eye growth were discussed. We have optimized a workable protocol for the study of the differential retinal protein expressions in the LIM using 2D-DIGE approach which was shown to have a number of advantages over the traditional 2D electrophoresis technique.     

Identification of clinically relevant protein targets in prostate cancer with 2D-DIGE coupled mass spectrometry and systems biology network platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.     

2D-difference gel electrophoretic proteomic analysis of a cell culture model of alveolar rhabdomyosarcoma

To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2-dimensional-difference gel electrophoresis (2D-DIGE), we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 nonredundant proteins. 2D-DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 nonredundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by Western blot analysis and immunohistochemistry (IHC). Thus, the 2D-DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies.     

Identification of the phosphotyrosine proteome from thrombin activated platelets

Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.     

Stage-related alterations in renal cell carcinoma--comprehensive quantitative analysis by 2D-DIGE and protein network analysis

Renal cell carcinoma accounts for about 3% of adult malignancies and 85% of neoplasms arising from the kidney. To identify potential progression markers for kidney cancer we examined non-neoplastic and neoplastic kidney tissue from three groups of patients, which represent different tumor stages (pT1, pT2, pT3) by a fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) approach combined with MALDI-ToF-MS/MS. Delta2D software package was used for gel image based quantification and statistical analysis. Thereby, a comprehensive Principal Component Analysis (PCA) could be performed and allowed a robust quality control of the experiment as well as a classification of the analyzed samples, which correlated with the predicted stages from the pathological examination. Additionally for selected candidate proteins we detected a correlation to the tumor grading as revealed by immunohistochemistry. On the 2D protein map 176 spots out of 989 were detected as at least 2-fold differentially expressed. These spots were analyzed by MALDI-ToF-MS/MS and 187 different proteins were identified. The functional clustering of the identified proteins revealed ten groups. Within these groups we found 86 enzymes, 63 proteins of unknown function, 14 transporter, 8 peptidases and 7 kinases. From the systems biology approach we could map many of these proteins in major pathways involved in remodelling of cytoskeleton, mitochondrial dysfunctions and changes in lipid metabolism. Due to complexity of the highly interconnected pathway network, further expression and functional validation of these proteins might provide new insights in kidney cancer progression to design novel diagnostic and therapeutic strategies.     

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.     

GM-CSF and TNFα modulate protein expression of human neutrophils visualized by fluorescence two-dimensional difference gel electrophoresis

Increased serum levels of TNFα and GM-CSF are found in various chronic inflammatory diseases and these cytokines affect the function of circulating and tissue neutrophils. TNFα- and GM-CSF-induced protein expression profiles could, therefore, serve as biomarker for the action of these cytokines in vivo. We stimulated human peripheral neutrophils with TNFα and GM-CSF in vitro and analyzed changes in their proteome by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). We report the differential expression of 3 and 18 protein spots following TNFα and GM-CSF stimulation, respectively. Differences in protein expression induced by TNFα were limited and did not show discriminatory power in a principal component analysis, whereas the profile induced by GM-CSF did. TNFα- and GM-CSF-induced both de novo IL-1β and sIL-1Ra protein expression as detected by Western blot analysis, which confirmed proper neutrophil activation by these cytokines in vitro. Mass spectrometry analysis of cytokine-regulated protein spots resulted in the identification of 8 proteins. Among the identified proteins, enolase 1 and annexin A1 might function as markers for peripheral neutrophil activation.In conclusion, a proteomic analysis of neutrophils by 2D-DIGE provides proof-of-principle that cytokine-induced protein profiles can serve as biomarkers for the action of individual cytokines in vivo.     

Proteome characterization of human NK-92 cells identifies novel IFN-alpha and IL-15 target genes

Natural killer (NK) cells are important components of innate immune defense. NK cells kill virus-infected cells and secrete cytokines that are involved in activation of other immune cells. Macrophage-derived cytokines interferon-alpha (IFN-alpha) and interleukin-15 (IL-15) are in turn important activators of NK cells, but the receptors and intracellular pathways that are involved in NK cell functions are still incompletely known. Here we have used expression proteomics to find new IFN-alpha and IL-15 regulated proteins in human NK-92 cells, which have the characteristics of activated NK cells. Cells were stimulated with cytokines for 20 h, lysed, and soluble proteins were separated by two-dimensional electrophoresis, and differentially expressed protein spots were identified with mass spectrometry and database searches. A total of 57 protein spots were found to be reproducibly differentially expressed between control and cytokine stimulated gel pairs, 26 spots being more than 2-fold upregulated and 3 spots being at least 2-fold downregulated. The rest 28 spots showed minor, less than 2-fold changes in their expression levels after quantification. From the differentially expressed protein spots we identified 47 different proteins, most of which are new IFN-alpha and IL-15 target proteins. Interestingly, we show that e.g., adenylate kinase 2 is highly upregulated by IFN-alpha and IL-15 stimulation in NK-92 cells. The expression of selected genes with high expression level differences after cytokine stimulation were further studied at mRNA level. Northern blot analysis showed that the genes studied were induced by IFN-alpha, IL-15, and IL-2 already at 3 h time point, suggesting that they are primary target genes of these cytokines.     

The effect of selenium enrichment on baker's yeast proteome

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE)-tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p ≤ 0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially was verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.     

The role of pseudokinases in cancer

Kinases play a critical role in regulating many cellular functions including development, differentiation and proliferation. To date, over 518 proteins with kinase activity, comprising ~2-3% of total cellular proteins, have been identified from within the human kinome. Interestingly, approximately 10% of kinases are categorised as pseudokinases since they lack one or more conserved catalytic residues within their kinase domain and were originally thought to have no enzymatic activity. Recently, there has been strong evidence to suggest that some pseudokinsases can not only function as scaffold proteins, but may also possess kinase activity leading to modulation of cell signalling pathways. Altered activity of these pseudokinases can result in impaired cellular function, particularly in malignancies. In this review we are discussing recent evidence that apart from a scaffolding role, pseudokinases also orchestrate cellular processes as active kinases per se in signalling pathways of malignant cells.     

Proteomics Approaches for Identification of Tumor Relevant Protein Targets in Pulmonary Squamous Cell Carcinoma by 2D-DIGE-MS

Potential markers for progression of pulmonary squamous cell carcinoma (SCC) were identified by examining samples of lung SCC and adjacent normal tissues using a combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF). The PANTHER System was used for gel image based quantification and statistical analysis. An analysis of proteomic data revealed that 323 protein spots showed significantly different levels of expression (P≤0.05) in lung SCC tissue compared to expression in normal lung tissue. A further analysis of these protein spots by MALDI-TOF-MS identified 81 different proteins. A systems biology approach was used to map these proteins to major pathways involved in numerous cellular processes, including localization, transport, cellular component organization, apoptosis, and reproduction. Additionally, the expression of several proteins in lung SCC and normal tissues was examined using immunohistochemistry and western blot. The functions of individual proteins are being further investigated and validated, and the results might provide new insights into the mechanism of lung SCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.     

Two-dimensional differential in gel electrophoresis (2D-DIGE) analysis of grape berry proteome during postharvest withering

The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement.     

Proteomic characterization of seeds from yellow lupin (Lupinus luteus L.)

Yellow lupin (Lupinus luteus L.) is a legume crop containing a large amount of protein in its seeds. In this study, we constructed a seed‐protein catalog to provide a foundation for further study of the seeds. A total of 736 proteins were identified in 341 2DE spots by nano‐LC‐MS/MS. Eight storage proteins were found as multiple spots in the 2DE gels. The 736 proteins correspond to 152 unique proteins as shown by UniRef50 clustering. Sixty‐seven of the 152 proteins were associated with KEGG‐defined pathways. Of the remaining proteins, 57 were classified according to a GO term. The functions of the remaining 28 proteins have yet to be determined. This is the first yellow lupin seed–protein catalog, and it contains considerably more data than previously reported for white lupin (L. albus L.).     

LCM纯化的鼻咽癌间质和正常鼻咽间质的定量蛋白质组学研究

间质在肿瘤发生发展中的作用越来越受到重视．为寻找与鼻咽癌( nasopharyngeal carcinoma,NPC )发生发展相关的特异性间质蛋白,采用激光捕获显微切割技术( laser capture microdissection,LCM )纯化鼻咽癌间质和正常鼻咽黏膜间质,荧光差异双向凝胶电泳( fluorescent two-dimensional difference gel electrophoresis 2-D,DIGE )结合质谱技术分离鉴定间质相关蛋白．Western blot及免疫组织化学技术验证了其中3个差异蛋白(CapG、L-plastin和S100A9),证实了2D-DIGE结果的可靠性．建立了LCM 纯化的鼻咽癌间质和正常鼻咽间质的荧光差异蛋白表达图谱,高通量筛选与肿瘤发生相关的间质蛋白,共得到34个有统计学意义的蛋白质点,质谱鉴定得到20个差异蛋白．研究结果提示：这些差异表达的蛋白质将有助于阐明鼻咽癌细胞和周围间质的关系．对间质蛋白功能的进一步研究,将有助于解析间质在肿瘤发生中的作用机制,并为从间质途径寻找肿瘤治疗靶标提供新思路．     

Lipid rafts are critical membrane domains in blood platelet activation processes

Among the various hematopoi;etic cells, platelets are critical for maintaining the integrity of the vascular system. They must be rapidly activated by sequential and coordinated mechanisms in order to efficiently prevent haemorrhage upon vascular injury. Several signal transduction pathways lead to platelet activation in vitro and in vivo, among them, several are initiated via receptors or co-receptors containing immuno-receptor tyrosine-based activation motifs (ITAM) which trigger downstream signalling like the immune receptors in lymphocytes. However, in contrast to immune cells for which the role of lipid rafts in signalling has largely been described, the involvement of laterally segregated membrane microdomains in platelet activation has been investigated only recently. The results obtained until now strongly suggest that early steps of platelet activation via the collagen receptor GpVI or via FcgammaRIIa occur preferentially in these microdomains where specific proteins efficiently organize key downstream signalling pathways. In addition, lipid rafts also contribute to platelet activation via heterotrimeric G-protein-coupled receptors. They are sites where the phosphoinositide (PI) metabolism is highly active, leading to a local generation of lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate. Here, evidence is accumulating that cholesterol-enriched membrane microdomains are part of a general process that contributes to the efficiency and the coordination of platelet activation mechanisms. Here we will discuss the biochemical and functional characterizations of human platelet rafts and their potential impact in platelet physiopathology.