Two-step OFFGEL approach for effective peptide separation compatible with iTRAQ labeling

Shotgun proteomic analyses are increasingly becoming methods of choice for complex samples. The development of effective methods for fractionating peptides to reduce the complexity of the sample before mass analysis is a key point in this strategy. The OFFGEL technology has recently become a tool of choice in proteomic analysis at peptide level. This OFFGEL electrophoresis (OGE) approach allows the in-solution separation of peptides from various biological sources by isoelectric focusing in highly resolved 24 fractions. It was also demonstrated that OGE technology is a filtering tool for pI-based validation of peptide identification. As peptide OGE is compatible with iTRAQ labeling, OGE is finding valuable applications in quantitative proteomics as well. The aim of this study is to explain a new 2D-OGE approach that improves the proteomic coverage of complex mixtures such as colorectal cell line lysates, and which is compatible with iTRAQ labeling

Visualisation tool for peptide fractionation data in proteomics: application to OFFGEL isoelectric focussing

<p>Abstract</p> <p>Background</p> <p>OFFGEL isoelectric focussing (IEF) has become a popular tool in proteomics to fractionate peptides or proteins. As a consequence there is a need for software solutions supporting data mining, interpretation and characterisation of experimental quality.</p> <p>Results</p> <p>We can assess performance characteristics of OFFGEL IEF peptide fractionation in proteomics by generating plots of the overall fractionation patterns and the pairwise comparisons of adjacent fractions.</p> <p>Conclusions</p> <p>A visualisation tool for peptide fractionation has been developed to support the evaluation of IEF data quality and can be implemented in proteomics research.</p

Modification of the secretion pattern of proteases, inflammatory mediators, and extracellular matrix proteins by human aortic valve is key in severe aortic stenosis

One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention

Le secretome, un modèle adapté à l'étude des protéines sécrétées par les tumeurs.<br />Application à l'analyse du rôle joué par p53 sur la sécrétion de protéines par des cellules du cancer du poumon.

Cancer is a real public health problem and will represent in 2010 the first cause of mortality. So the knowledge of this disease and its mechanisms remains a great challenge. Malignant processes such as metastasis, invasion, or angiogenesis are tightly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. The mutation of pro- and anti-oncogenic factors plays an important role in tumor progression. In this work, we want to ask the following major question: is it interesting to study by proteomics the secreted proteins by tumor to better understand tumorigenesis mechanisms and is it useful for clinical biomarker discovery? We focused on lung cancer which is the cancer with the greater mortality and incidency frequences in the world. This cancer has the greater p53 mutation rates. This protein is a major tumor suppressor known to modulate the release of proteins by the tumor cells; however, while p53-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. Here, we developed a proteomics process using iTRAQ labeling, OFFGEL isoelectric focusing and LC-MALDI-MS/MS analysis efficient to the study of this class of proteins. We used it to characterize the p53-dependent secretome of a lung tumor model in vitro. We identified 909 proteins released by these cells, among which 91 are p53-modulated. This differential secretome analysis promises more detailed of how the loss of wt-p53 contributes to tumorigenic processes through the modulation of secreted proteins. We also demonstrated that the modulation of exported proteins can be detected in vivo in total protein extracts and plasma of tumor-bearing mice. This is the first report establishing that the in vitro cell line secretome is reliable and reflects the extracellular release of proteins from tumor cells in vivo and could be used to identify putative tumor markers.Le cancer est un véritable problème de santé publique et représentera en 2010 la première cause de mortalité dans le monde. La lutte contre cette maladie est donc, plus que jamais, un enjeu capital. Les processus tumoraux associés à la carcinogénèse, tels que la métastase, l'invasion ou l'angiogenèse, dépendent étroitement de la composition du milieu extracellulaire, lui-même affecté par la sécrétion de protéines par les cellules tumorales. La mutation ou la variation d'expression de facteurs pro- ou anti-oncogénique joue un rôle important dans le développement et la progression de la tumeur. Durant ce travail de thèse, nous avons voulu apporter des éléments de réponse à la problématique suivante : l'étude par protéomique des protéines sécrétées par la tumeur apporte t'elle des informations contribuant à la compréhension des mécanismes de la tumorogenèse et peut elle permettre de mettre en valeur de nouvelles cibles d'intérêt clinique ? Nous avons ciblé notre étude sur le cancer du poumon, le plus fréquent au niveau mondial tant en terme d'incidence que de mortalité. Ce cancer présente le plus fort taux de mutation du gène p53, un anti-oncogène clé de la cellule. Cette perte de fonction module la sécrétion de nombreuses protéines dont l'investigation est primordiale, bien que paradoxalement, peu étudiée. Notre travail s'est ainsi focalisé sur l'étude de l'influence de p53 sur la modulation de la sécrétion de protéines par les tumeurs du poumon. Nous avons ainsi développé un procédé d'analyse protéomique adapté, basé notamment sur un marquage iTRAQ, une séparation par isoélectrofocalisation de type OFFGEL et une analyse LC-MALDI-MS/MS. Ce procédé a été appliqué à l'étude du rôle joué par l'expression conditionnelle de p53 sur la modulation du secretome d'une lignée cellulaire d'adénocarcinome du cancer du poumon non à petites cellules. Plusieurs protéines d'intérêt ont ainsi été caractérisées et confirmées in vivo chez la souris au niveau tumoral et plasmatique. Ces résultats apportent une meilleure compréhension du rôle primordial joué par l'altération de p53 dans la modulation de l'environnement tumoral et font des secretomes cellulaires un modèle de choix pour l'identification de marqueurs tumoraux

Le secretome, un modèle adapté à l'étude des protéines sécrétées par les tumeurs (application à l'analyse du rôle joué par p53 sur la sécrétion de protéines par des celllules du cancer du poumon)

Le cancer est un véritable problème de santé publique et représentera en 2010 la première cause de mortalité dans le monde. La lutte contre cette maladie est donc, plus que jamais, un enjeu capital. Les processus tumoraux associés à la carcinogénèse, tels que la métastase, l'invasion ou l'angiogenèse, dépendent étroitement de la composition du milieu extracellulaire, lui-même affecté par la sécrétion de protéines par les cellules tumorales. La mutation ou la variation d'expression de facteurs pro- ou anti-oncogénique joue un rôle important dans le développement et la progression de la tumeur. Durant ce travail de thèse, nous avons voulu apporter des éléments de réponse à la problématique suivante : l'étude par protéomique des protéines sécrétées par la tumeur apporte t'elle des informations contribuant à la compréhension des mécanismes de la tumorogenèse et peut elle permettre de mettre en valeur de nouvelles cibles d'intérêt clinique ? Nous avons ciblé notre étude sur le cancer du poumon, le plus fréquent au niveau mondial tant en terme d'incidence que de mortalité. Ce cancer présente le plus fort taux de mutation du gène p53, un anti-oncogène clé de la cellule. Cette perte de fonction module la sécrétion de nombreuses protéines dont l'investigation est primordiale, bien que paradoxalement, peu étudiée. Notre travail s'est ainsi focalisé sur l'étude de l'influence de p53 sur la modulation de la sécrétion de protéines par les tumeurs du poumon. Nous avons ainsi développé un procédé d'analyse protéomique adapté, basé notamment sur un marquage iTRAQ, une séparation par isoélectrofocalisation de type OFFGEL et une analyse LC-MALDI-MS/MS. Ce procédé a été appliqué à l'étude du rôle joué par l'expression conditionnelle de p53 sur la modulation du secretome d'une lignée cellulaire d'adénocarcinome du cancer du poumon non à petites cellules. Plusieurs protéines d'intérêt ont ainsi été caractérisées et confirmées in vivo chez la souris au niveau tumoral et plasmatique. Ces résultats apportent une meilleure compréhension du rôle primordial joué par l'altération de p53 dans la modulation de l'environnement tumoral et font des secretomes cellulaires un modèle de choix pour l'identification de marqueurs tumoraux.Cancer is a real public health problem and will represent in 2010 the first cause of mortality. So the knowledge of this disease and its mechanisms remains a great challenge. Malignant processes such as metastasis, invasion, or angiogenesis are tightly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. The mutation of pro- and anti-oncogenic factors plays an important role in tumor progression. In this work, we want to ask the following major question: is it interesting to study by proteomics the secreted proteins by tumor to better understand tumorigenesis mechanisms and is it useful for clinical biomarker discovery? We focused on lung cancer which is the cancer with the greater mortality and incidency frequences in the world. This cancer has the greater p53 mutation rates. This protein is a major tumor suppressor known to modulate the release of proteins by the tumor cells; however, while p53-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. Here, we developed a proteomics process using iTRAQ labeling, OFFGEL isoelectric focusing and LC-MALDI-MS/MS analysis efficient to the study of this class of proteins. We used it to characterize the p53-dependent secretome of a lung tumor model in vitro. We identified 909 proteins released by these cells, among which 91 are p53-modulated. This differential secretome analysis promises more detailed of how the loss of wt-p53 contributes to tumorigenic processes through the modulation of secreted proteins. We also demonstrated that the modulation of exported proteins can be detected in vivo in total protein extracts and plasma of tumor-bearing mice. This is the first report establishing that the in vitro cell line secretome is reliable and reflects the extracellular release of proteins from tumor cells in vivo and could be used to identify putative tumor markers.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

[Secretome: definitions and biomedical interest]

International audienceINTRODUCTION: The secretome, or secretomics refers to the global study of proteins that are secreted by a cell, a tissue or an organism. EXEGESIS: The secretome is an important class of proteins that control many biological and physiological processes. Many secretion pathways are implicated in the release of these proteins. CONCLUSION: The secretome is a potential source suitable for the discovery of new therapeutic targets or biomarker candidates

Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling.

International audienceABSTRACT: BACKGROUND: The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability necessary for the analysis of such complex proteomes. RESULTS: We evaluated OFFGEL fractionator technology to separate peptides from two complex proteomes, human secretome and human plasma, using a 24-wells device encompassing the pH range 3-10. In combination with reverse phase liquid chromatography, peptides from these two samples were separated and identified by MALDI TOF-TOF. The repartition profiles of the peptides in the different fractions were analyzed and explained by their content in charged amino acids using an algorithmic model based on the possible combinations of amino acids. We also demonstrated for the first time the compatibility of OFFGEL separation technology with the quantitative proteomic labeling technique iTRAQ allowing inclusion of this technique in complex samples comparative proteomic workflow. CONCLUSION: The reported data showed that OFFGEL system provides a highly valuable tool to fractionate peptides from complex eukaryotic proteomes (plasma and secretome) and is compatible with iTRAQ labeling quantitative studies. We therefore consider peptides OFFGEL fractionation as an effective addition to our strategy and an important system for quantitative proteomics studies