Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry

Completion of the human genome sequence has provided scientists with powerful resources with which to explore the molecular events associated with disease states such as diabetes. Understanding the relative levels of expression of gene products, especially of proteins, and their post-translational modifications will be critical. However, though the pancreatic islets play a key role in glucose homeostasis, global protein expression data in human are decidedly lacking. We here report the two-dimensional protein map and database of human pancreatic islets. A high level of reproducibility was obtained among the gels and a total of 744 protein spots were detected. We have successfully identified 130 spots corresponding to 66 different protein entries and generated a reference map of human islets. The functionally characterized proteins include enzymes, chaperones, cellular structural proteins, cellular defense proteins, signaling molecules, and transport proteins. A number of proteins identified in this study (e.g., annexin A2, elongation factor 1-alpha 2, histone H2B.a/g/k, heat shock protein 90 beta, heat shock 27 kDa protein, cyclophilin B, peroxiredoxin 4, cytokeratins 7, 18, and 19) have not been previously described in the database of mouse pancreatic islets. In addition, altered expression of several proteins, like GRP78, GRP94, PDI, calreticulin, annexin, cytokeratins, profilin, heat shock proteins, and ORP150 have been associated with the development of diabetes. The data presented in this study provides a first-draft reference map of the human islet proteome, that will pave the way for further proteome analysis of pancreatic islets in both healthy and diabetic individuals, generating insights into the pathophysiology of this condition.     

The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells. In 38 cases we observed an increase and in 11 cases a decrease in content in the sickle cell membranes. The proteins of interest were identified by in-gel tryptic digestion followed by liquid chromatography in line with tandem mass spectrometry. From 38 analyzed gel spots, we identified 44 protein forms representing different modifications of 22 original protein sequences. The majority of the identified proteins fall into small groups of related proteins of the following five categories: actin accessory proteins--four proteins, components of lipid rafts--two proteins, scavengers of oxygen radicals--two proteins, protein repair participants--six proteins, and protein turnover components--three proteins. The number of proteins whose content in sickle RBC membrane is decreased is noticeably smaller, and most are either components of lipid rafts or actin accessory proteins. Elevated content of protein repair participants as well as oxygen radical scavengers may reflect the increased oxidative stress observed in sickle cells.     

Yeast proteome map (update 2006)

To improve the potential of two-dimensional gel electrophoresis for proteomic investigations in yeast we have undertaken the systematic identification of Saccharomyces cerevisiae proteins separated on 2-D gels. We report here the identification of 187 novel protein spots. They were identified by two methods, mass spectrometry and gene inactivation. These identifications extend the number of protein spots identified on our yeast 2-D proteome map to 602, i.e. nearly half the detectable spots of the proteome map. These spots correspond to 417 different proteins. The reference map and the list of identified proteins can be accessed on the Yeast Protein Map server (www.ibgc.u-bordeaux2.fr/YPM).     

Differential expression of equine muscle biopsy proteins during normal training and intensified training in young standardbred horses using proteomics technology

The major aim of the present study was to investigate the proteome of standardbred horses at different stages of training and intensified training. We searched for biomarkers using small skeletal muscle biopsies of live animals. 2D gel electrophoresis and mass spectrometry were successfully applied to investigate training-induced differential expression of equine muscle biopsy proteins. Despite the poor resolution of the equine genome and proteome, we were able to identify the proteins of 20 differential spots representing 16 different proteins. Evaluation of those proteins complies with adaptation of the skeletal muscle after normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain fatty acids, and to store energy in the form of glycogen. Intensified training leads to additional changed spots. Alpha-1-antitrypsin was found increased after intensified training but not after normal training. This protein may thus be considered as a marker for overtraining in horses and also linked to overtraining in human athletes.     

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

Background

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.

Principal Findings

Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions.

Conclusions

The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.     

The Fas-induced apoptosis analyzed by high throughput proteome analysis

The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect to de novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1alpha became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo.     

New protein clustering of breast cancer tissue proteomics using actin content as a cellularity indicator

In the present study, we report the comparative proteome profiles of proteins solubilized from 37 breast cancer surgical tissues, normalized for the actin content. Blood-derived proteins were excluded from the analysis. Among the tumor-derived protein spots, a large proportion (39%) was found present in all patients. These included several glycolytic enzymes, detox and heat shock proteins, members of annexin and S100 protein families, cathepsin D, and two "rare" proteins, DDAH2 involved in the angiogenesis control, and the oncogene PARK7. Other proteins, such as psoriasin, galectin1, cofilin, peroredoxins, SH3L1, and others, showed sporadic presence and high expression level, which suggests their possible role for patient stratification.     

Proteomic profiling reveals compartment-specific, novel functions of ascidian sperm proteins

In this study, we performed extensive proteomic analysis of sperm from the ascidian Ciona intestinalis. Sperm were fractionated into heads and flagella, followed by further separation into Triton X-100-soluble and -insoluble fractions. Proteins from each fraction and whole sperm were separated by isoelectric focusing using two different pH ranges, followed by SDS-PAGE at two different polyacrylamide concentrations. In total, 1,294 protein spots representing 304 non-redundant proteins were identified by mass spectrometry (MALDI-TOF). On comparison of the proteins in each fraction, we were able to identify the proteins specific to different sperm compartments. Further comparison with the testis proteome allowed the pairing of proteins with sperm-specific functions. Together with information on gene expression in developing embryos and adult tissues, these results provide insight into novel cellular and functional aspects of sperm proteins, such as distinct localization of actin isoforms, novel Ca(2+)-binding proteins in axonemes, localization of testis-specific serine/threonine kinase, and the presence of G-protein coupled signaling and ubiquitin pathway in sperm flagella.     

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130±102 and 1200±97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35% of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels.     

Industrial process proteomics: alfalfa protein patterns during wet fractionation processing

A proteome reference map of major soluble proteins from Medicago sativa (alfalfa) leaves and stems has been established for the first time. Among 195 spots analyzed by mass spectrometry and N-terminal Edman sequencing, 117 spots were unambiguously identified, representing 87 different proteins. Of these 87 proteins, 13 proteins were directly identified from the partial genome of Medicago sativa, 30 from expressed sequenced tags (ESTs) of the model legume Medicago truncatula and 44 from closely relative species by a cross-species protein identification method. The proteome map of Medicago sativa was then set as a reference to study the major high protein content products that are generated during the wet fractionation process of alfalfa green biomass. Using two-dimensional electrophoresis, we studied the variation of the protein patterns at different steps of the industrial-scale process. We clearly show that the process induces significant changes including chemical modifications, proteolytic events, and heat-shock protein responses. Strikingly, a certain level of cellular regulation is conserved during biomass processing, as exemplified by the induction of some heat shock proteins. Finally, all the results obtained in this proteomic study may help to identify novel products and to improve process designs in alfalfa biomass plants.     

Actin deficiency induces cofilin phosphorylation: proteome analysis of HeLa cells after beta-actin gene silencing

Actin-binding proteins regulate the dynamic structure and function of actin filaments in the cell. Much is known about how manipulation of the actin-binding proteins affects the structure and function of actin filaments; however, little is known about how manipulation of actin in the cell affects actin-binding proteins. We addressed this question by utilizing two technologies: RNA interference and 2-dimensional gel electrophoresis. We knocked down beta-actin expression in HeLa cells using short interfering RNA and applied 2-DGE to examine alterations in the HeLa cell proteome. We revealed a 2-5 fold increases of four protein spots on 2-D gels and identified these proteins by mass spectrometry. Three of the four proteins were actin-binding proteins, including cofilin, which promotes both disassembly and assembly of actin filaments but becomes inactivated when phosphorylated. Further examination revealed that the cofilin total protein level barely increased, but the phosphorylated cofilin level increased dramatically in HeLa cells after beta-actin siRNA treatment. These results suggest that in response to siRNA-induced beta-actin deficiency HeLa cells inactivate cofilin by phosphorylation rather than down-regulate its protein expression level. This study also demonstrates that the combination of RNA interference and 2-dimensional gel electrophoresis technologies provides a valuable method to study protein interactions in a specific cellular pathway.     

A proteomics approach to the study of the molecular consequence of IgE-mediated cell signalling in RBL-2H3.1 cells and 2D reference map preparation for the RBL-2H3.1 cell line

A highly reproducible 2D (two-dimensional) map for the proteome and a pattern of protein phosphorylation of high secretory variant of RBL-2H3 cells (RBL-2H3.1) (a model cell in allergy studies) in resting and treated cells with IgE or IgE+Ag are presented. Major molecular changes were seen in the proteome of 3 h-activated cells with IgE+Ag, especially for proteins of ～17 kDa compared with the control. We have identified 13 proteins on 11 corresponding spots as up-regulated proteins in response to IgE+Ag activation. Also, protein identification on 55 spots with MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) and ESI-MS (electrospray ionization mass spectrometry) resulted in a reliable 2D reference map and an opportunity for the subsequent use of a 1 min-activated cell map for a phosphoproteomics study.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

Storage‐induced changes of the cytosolic red blood cell proteome analyzed by 2D DIGE and high‐resolution/high‐accuracy MS

The storage of packed red blood cells (RBCs) is associated with the development of morphological and biochemical changes leading to a reduced posttransfusion functionality and viability of the cells. Within this study, 2D DIGE and high‐resolution/high‐accuracy Orbitrap MS were used to analyze the storage‐induced changes of the cytosolic RBC proteome and identify characteristic protein patterns and potential marker proteins for the assessment of RBC storage lesions. Leukodepleted RBC concentrates were stored according to standard blood bank conditions for 0, 7, 14, 28, and 42 days and analyzed by using a characterized and validated protocol. Following statistical evaluation, a total of 14 protein spots were found to be significantly altered after 42 days of ex vivo storage. Protein identification was accomplished by tryptic digestion and LC‐MS/MS and three proteins potentially useful as biomarkers for RBC aging comprising transglutaminase 2, beta actin, and copper chaperone for superoxide dismutase were selected and validated by western blot analysis. These can serve as a basis for the development of a screening assay to detect RBC storage lesions and autologous blood doping in sports.     

Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool

The combination of laser microdissection and two-dimensional gel electrophoresis (2-D PAGE) has been developed to perform proteomic analysis on specific populations of cells in cancer tissues. However, as conventional low sensitivity silver staining was used for spot detection, the microdissection required to obtain an adequate amount of protein for 2-D PAGE is laborious and only a restricted number of protein spots could be visualized. As a consequence, this technology was impractical for direct clinical applications and had a limited impact on cancer studies. To solve these problems, we developed an application in which fluorescent dyes label the proteins extracted from microdissected tissues prior to 2-D PAGE separation. In this application, a small amount of protein, less than 6.6 microg, was enough to generate a 2-D profile with approximately 1500 protein spots. This technique was applied to compare the proteome of normal intestinal epithelium with that of adenoma in Min mice. Thirty-seven protein spots reproducibly showed significant differences in intensities. Mass spectrometric analysis and Western blotting identified eight of them, including prohibitin, 14-3-3zeta, tropomyosin 3 and Hsp84. These results indicate that fluorescence labeling of proteins from microdissected tissues prior to 2-D PAGE is a powerful cancer proteomic study tool.     

Comparative proteomic analysis of human pancreatic juice: methodological study

Pancreatic cancer is the most lethal of all the common malignancies. Markers for early detection of this disease are urgently needed. Here, we optimized and applied a proteome analysis of human pancreatic juice to identify biomarkers for pancreatic cancer. Pancreatic juice samples, devoid of blood or bile contamination, were collected from patients with pancreatic cancer (n = 5), benign pancreatic diseases (n = 6), or cholelithiasis (n = 3) during endoscopic retrograde cholangiopancreatography (ERCP). After ultramembrane centrifugation sample preparation, pancreatic juice proteins were separated by 2-DE and identified by MALDI-TOF-MS. A 2-DE dataset of pancreatic juice from patients with cholelithiasis was established, consisting of 76 protein spots representing 22 different proteins. Disease-associated obstruction of the pancreatic duct strongly effected the protein composition of pancreatic juice. Concurrently, pancreatic juice from patients with pancreatic cancer was compared to nonmalignant controls with comparable obstruction of pancreatic ducts. Seven protein spots were identified that consistently appeared at changed levels in pancreatic juice from patients with pancreatic cancer. In conclusion, comparative proteomic analysis of human pancreatic juice can be used to identify biomarkers of pancreatic cancer.     

Mapping the proteome of Leishmania Viannia parasites using two-dimensional polyacrylamide gel electrophoresis and associated technologies

In this study we have demonstrated the potential of two-dimensional electrophoresis (2DE)-based technologies as tools for characterization of the Leishmania proteome (the expressed protein complement of the genome). Standardized neutral range (pH 5-7) proteome maps of Leishmania (Viannia) guyanensis and Leishmania (Viannia) panamensis promastigotes were reproducibly generated by 2DE of soluble parasite extracts, which were prepared using lysis buffer containing urea and nonidet P-40 detergent. The Coomassie blue and silver nitrate staining systems both yielded good resolution and representation of protein spots, enabling the detection of approximately 800 and 1,500 distinct proteins, respectively. Several reference protein spots common to the proteomes of all parasite species/strains studied were isolated and identified by peptide mass spectrometry (LC-ES-MS/MS), and bioinformatics approaches as members of the heat shock protein family, ribosomal protein S12, kinetoplast membrane protein 11 and a hypothetical Leishmania-specific 13 kDa protein of unknown function. Immunoblotting of Leishmania protein maps using a monoclonal antibody resulted in the specific detection of the 81.4 kDa and 77.5 kDa subunits of paraflagellar rod proteins 1 and 2, respectively. Moreover, differences in protein expression profiles between distinct parasite clones were reproducibly detected through comparative proteome analyses of paired maps using image analysis software. These data illustrate the resolving power of 2DE-based proteome analysis. The production and basic characterization of good quality Leishmania proteome maps provides an essential first step towards comparative protein expression studies aimed at identifying the molecular determinants of parasite drug resistance and virulence, as well as discovering new drug and vaccine targets.     

Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.