Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation

<p>Abstract</p> <p>Background</p> <p>The development of mass spectrometric techniques and fractionation methods now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Nevertheless, this work is particularly difficult due to the wide dynamic range of protein concentration in eukaryotic tissues. In this paper, we present a shotgun method whereby the peptides are fractionated using OFFGEL electrophoresis after iTRAQ labelling.</p> <p>Results</p> <p>We demonstrated that iTRAQ peptide labelling enhances MALDI ionisation and that the OFFGEL fractionation of the labelled peptides introduces a supplementary criterion (pI) useful for validation and identification of proteins. We showed that iTRAQ samples allowed lower-concentrated proteins identification in comparison with free-labelled samples.</p> <p>Conclusion</p> <p>The combined use of iTRAQ labelling and OFFGEL fractionation allows a considerable increase in proteome coverage of very complex samples prepared from total cell extracts and supports the low-concentrated protein identification.</p

A Proteomic Approach for Plasma Biomarker Discovery with iTRAQ Labelling and OFFGEL Fractionation

Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma “signatures,” which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 μg of plasma proteins

Recherche de biomarqueurs de la neurotoxicité des traitements anticancéreux à base d'oxaliplatine: approche protéomique quantitative

Oxaliplatin is a reference drug in the treament of metastatic colorectal cancer. However Oxaliplatin causes a neurological toxicity which alters the quality of life of some predisposed patients. We analysed for the first ti me oxaliplatin neurotoxicity by a proteomic approach. An experimental protocol associating iTRAQ labelling and OFFGEL-IEF fractionation was developed in order to improve the proteome coverage of complex samples. Then, the differential protein expression following a clinically relevant oxaliplatin treatment was globally analysed both in the intracellular proteome and in the secretome using a human cellular mode1 for neurotoxicity. The intracellular proteome analysis allowed the identification of more than 2700 proteins and offers new perspectives into the molecular mechanisms of oxaliplatin neurotoxicity. Oxaliplatin alters the expression of proteins involved in DNA damage response, cell cycle control, oxidative stress, energy metabolism, proteotoxic stress, multidrug resistance, neuronal plasticity and calcium homeostasis. The secretome analysis exhibited 23 over-expressed secreted proteins following oxaliplatin treatment. We suggest for the first time that Calmodulin,Thymosin beta-10 and the neurotrophic factor Neudesin could be potentiaI biomarkers for oxaliplatin neuronal injuy.L'oxaliplatine est un médicament de référence dans les traitements des cancers colorectaux métastatiques. Toutefois, l'oxaliplatine occasionne une toxicité d'ordre neurologique qui retentit sur la qualité de vie de certains patients prédisposés. Nous avons analysé pour la première fois la neurotoxicité de l'oxaliplatine par une approche protéomique. Un protocole expérimental, associant marquage iTRAQ et fractionnement par IEFOFFGEL a tout d'abord été mis en place afin d'améliorer la couverture protéomique d'échantillons complexes. Puis, l'expression protéique différentielle après traitement par I'oxaliplatine a été analysée globalement, à la fois dans le protéome intracellulaire et dans le sécrétome d'un modèle cellulaire humain de nerotoxicité. L'analyse du protéome intracellulaire a permis l'identification de plus de 2700 protéines et offre de nouvelles perspectives quant aux mécanismes moléculaires de la neurotoxicité de I ' o x aliplatine. Le médicament aItère l'expression de protéines impliquées dans la réponse aux dommages à l'ADN, le contrôle du cycle cellulaire, le stress oxydatif , le métabolisme énergétique, le stress protéotoxique, la résistance multidrogue, la plasticité neuronale et l'homéostasie calcique. L'analyse du sécrétome a mis en évidence la sur-expression, suite au traitement par l'oxaliplatine, de 23 protéines sécrétées. Nous proposons pour la première fois les protéines Calmoduline, Thymosine beta-10 et le facteur neurotrophique Neudésine comme candidats biomarqueurs de la neurotoxicité des traitements anticancéreux à base d'oxaliplatine

Recherche de biomarqueurs de la neurotoxicité des traitements anticancéreux à base d'oxaliplatine (approche protéomique quantitative)

L'oxaliplatine est un médicament de référence dans les traitements des cancers colorectaux métastatiques. Toutefois, l'oxaliplatine occasionne une toxicité d'ordre neurologique qui retentit sur la qualité de vie de certains patients prédisposés. Nous avons analysé pour la première fois la neurotoxicité de l'oxaliplatine par une approche protéomique. Un protocole expérimental, associant marquage iTRAQ et fractionnement par IEFOFFGEL a tout d'abord été mis en place afin d'améliorer la couverture protéomique d'échantillons complexes. Puis, l'expression protéique différentielle après traitement par I'oxaliplatine a été analysée globalement, à la fois dans le protéome intracellulaire et dans le sécrétome d'un modèle cellulaire humain de nerotoxicité. L'analyse du protéome intracellulaire a permis l'identification de plus de 2700 protéines et offre de nouvelles perspectives quant aux mécanismes moléculaires de la neurotoxicité de I ' o x aliplatine. Le médicament aItère l'expression de protéines impliquées dans la réponse aux dommages à l'ADN, le contrôle du cycle cellulaire, le stress oxydatif , le métabolisme énergétique, le stress protéotoxique, la résistance multidrogue, la plasticité neuronale et l'homéostasie calcique. L'analyse du sécrétome a mis en évidence la sur-expression, suite au traitement par l'oxaliplatine, de 23 protéines sécrétées. Nous proposons pour la première fois les protéines Calmoduline, Thymosine beta-10 et le facteur neurotrophique Neudésine comme candidats biomarqueurs de la neurotoxicité des traitements anticancéreux à base d'oxaliplatine.Oxaliplatin is a reference drug in the treament of metastatic colorectal cancer. However Oxaliplatin causes a neurological toxicity which alters the quality of life of some predisposed patients. We analysed for the first ti me oxaliplatin neurotoxicity by a proteomic approach. An experimental protocol associating iTRAQ labelling and OFFGEL-IEF fractionation was developed in order to improve the proteome coverage of complex samples. Then, the differential protein expression following a clinically relevant oxaliplatin treatment was globally analysed both in the intracellular proteome and in the secretome using a human cellular mode1 for neurotoxicity. The intracellular proteome analysis allowed the identification of more than 2700 proteins and offers new perspectives into the molecular mechanisms of oxaliplatin neurotoxicity. Oxaliplatin alters the expression of proteins involved in DNA damage response, cell cycle control, oxidative stress, energy metabolism, proteotoxic stress, multidrug resistance, neuronal plasticity and calcium homeostasis. The secretome analysis exhibited 23 over-expressed secreted proteins following oxaliplatin treatment. We suggest for the first time that Calmodulin,Thymosin beta-10 and the neurotrophic factor Neudesin could be potentiaI biomarkers for oxaliplatin neuronal injuy.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

A Nonsense Mutation in the ERG6 Gene Leads to Reduced Susceptibility to Polyenes in a Clinical Isolate of Candida glabrata▿

Unlike the molecular mechanisms that lead to azole drug resistance, the molecular mechanisms that lead to polyene resistance are poorly documented, especially in pathogenic yeasts. We investigated the molecular mechanisms responsible for the reduced susceptibility to polyenes of a clinical isolate of Candida glabrata. Sterol content was analyzed by gas-phase chromatography, and we determined the sequences and levels of expression of several genes involved in ergosterol biosynthesis. We also investigated the effects of the mutation harbored by this isolate on the morphology and ultrastructure of the cell, cell viability, and vitality and susceptibility to cell wall-perturbing agents. The isolate had a lower ergosterol content in its membranes than the wild type, and the lower ergosterol content was found to be associated with a nonsense mutation in the ERG6 gene and induction of the ergosterol biosynthesis pathway. Modifications of the cell wall were also seen, accompanied by increased susceptibility to cell wall-perturbing agents. Finally, this mutation, which resulted in a marked fitness cost, was associated with a higher rate of cell mortality. Wild-type properties were restored by complementation of the isolate with a centromeric plasmid containing a wild-type copy of the ERG6 gene. In conclusion, we have identified the molecular event responsible for decreased susceptibility to polyenes in a clinical isolate of C. glabrata. The nonsense mutation detected in the ERG6 gene of this isolate led to a decrease in ergosterol content. This isolate may constitute a useful tool for analysis of the relevance of protein trafficking in the phenomena of azole resistance and pseudohyphal growth