Aplicació de tècniques proteòmiques de quantificació i validació en recerca biomèdica

Les tècniques proteòmiques basades en l'espectrometria de masses (MS) permeten la caracterització de la complexa i dinàmica natura del proteoma. Aquestes eines han contribuit a entendre millor certes funcions biològiques i respostes cel·lulars i a obtenir biomarcadors específics per a l'estudi d'algunes malalties. En aquest treball, es van utilitzar tècniques proteòmiques de quantificació i validació, basades en l'espectrometria de masses (MS), en diferents estudis de recerca biomèdica. En el primer estudi es va comparar el perfil de proteïnes del fluid vitri de pacients diabètics amb retinopatia diabètica proliferativa (PDR) amb el de pacients no diabètics amb forat macular idiopàtic (MH). L'anàlisi proteòmica comparativa es va fer amb el sistema d'electroforesi diferencial en gel bidimensional basat en el marcatge fluorescent (2D-DIGE). Per MS es van identificar 11 proteïnes diferencials entre els pacients amb PDR i els individus no diabètics. Cinc de les proteïnes diferencials és van validar per western blot en fluids vitris i per RT-PCR en retines. L'anàlisi proteòmica 2D-DIGE va servir per a identificar potencials candidats involucrats en la patogènesis de la PDR. En el segon estudi es va estudiar l'efecte del gen REgulador AutoImmune (AIRE) mitjançant la comparació dels proteomes de cèl·lules epitelials transfectades o no amb AIRE. L'anàlisi comparativa es va realitzar combinant dues tècniques de proteòmica quantitativa: la 2D-DIGE i l'etiquetatge isotòpic codificat de proteïna (ICPL) en combinació amb cromatografia líquida acoblada a espectrometria de masses (LC-MS). Els resultats van mostrar un nivell incrementat de varies xaperones en les cèl·lules que expressaven AIRE, mentre que diferents proteïnes de interacció del citoesquelet es van trobar disminuïdes. A més, algunes proteïnes relacionades amb apoptosi tenien abundàncies diferencials entre unes cèl·lules i les altres. Aquests resultats es van confirmar per western blot i citometria de flux. Finalment, assajos d'apoptosi amb annexin V i etopòsid van demostrar que les cèl·lules positives en AIRE patien més apoptosi espontània i eren menys resistents a la inducció d'apoptosi. Els resultats obtinguts van corroborar el paper d'AIRE com a inductor d'apoptosi. En el tercer estudi, amb l'objectiu de identificar possibles proteïnes biomarcadores de l'activitat TGFβ en gliomes es van analitzar les proteïnes secretades en els cultius cel·lulars primaris derivats de tumors (PCTCs), tractats o no amb TGFβ, mitjançant experiments quantitatius de LC-MS sense marcatge i ICPL. Es van identificar varies proteïnes candidates per a les que es van desenvolupar mètodes d'anàlisi de seguiment d'una reacció seleccionada (SRM) per a poder validar-les en mostres de líquid cefaloraquidi (CSF) i plasma de pacients amb glioma. Per l'anàlisi dels CSFs els resultats van mostrar una clara correlació entre les proteïnes candidates i els nivells de TGFβ en aquestes mostres. Igualment, al analitzar les proteïnes candidates per SRM en mostres de plasma de pacients amb glioma en fases alternades de tractament amb un inhibidor de TGFβ es va observar que els nivells de les proteïnes d'interès eren modulades pel tractament. Aquestes proteïnes conformarien una firma proteica que podria ser útil per al diagnòstic i el seguiment del tractament dels pacients. En conjunt, els resultats obtinguts en cada un dels tres estudis demostren la utilitat de l'anàlisi proteòmica en diferents aspectes de la investigació biomèdicaProteomic techniques based on mass spectrometry (MS) allow the characterization of the complex and dynamic nature of the proteome. These tools have contributed to better understand certain biological functions and cellular responses and to obtain specific biomarkers useful for the management of some diseases. In this work, quantitative and validation proteomic techniques, based on mass spectrometry, were applied to different biomedical research projects. In the first study, the protein profile of the vitreous fluid of diabetic patients with proliferative diabetic retinopathy (PDR) and non diabetic patients with macular hole (MH) was compared. Comparative proteomic analysis was performed using differential in gel electrophoresis based on fluorescent labeling (2D_DIGE). Eleven differential proteins between PDR and non diabetic patients were identified by MS. Five differential proteins were validated by western blot analysis of vitreous fluid and by RT-PCR of retinas. 2D-DIGE proteomic analysis allowed the identification of potential candidates involved in the pathogenesis of PDR. In the second study, the effect of AutoImmune Regulator (AIRE) gen was studied by comparison of the proteomic profile of epithelial cells transfected or not with AIRE. The comparative analysis was done using to proteomic techniques: 2D-DIGE and Isotopic Coded Protein Labeling (ICPL) in combination with liquid chromatography coupled to mass spectrometry (LC-MS). Results showed an increased level of some chaperons in the cells expressing AIRE, while some cytoskeleton interacting proteins were decreased. Moreover, proteins related wit apoptosis had differential abundances between samples. These results were confirmed by western blot and flow cytometry. Finally, apoptosis assays with annexin V and etoposide demonstrated that AIRE-positive cells suffer more spontaneous apoptosis and are less resistant to apoptosis induction. These results confirm the role of AIRE as an inducer of apoptosis. In the third study, the main objective was the identification of biomarker proteins of TGFβ activity in gliomas. Secreted proteins from primary cultures of tumor cells (PCTCs), treated or not with TGFβ, were analyzed by label free and ICPL LC-MS quantitative experiments. Some candidate proteins were identified. Selected reaction monitoring (SRM) methods were designed to analyze these candidate proteins in cerebrospinal fluid (CSF) and plasma of glioma patients. CSF analysis showed a clear relationship between the protein levels and the TGFβ concentration. When the SRM methods were applied to the plasma of glioma patients under alternate periods of treatment with a TGFβ inhibitor, the levels of proteins of interest were observed to be modulated by the treatment. These proteins can thus constitute a protein signature useful for diagnostic and treatment monitoring. Overall, the results obtained in each of the projects show the usefulness of the proteomic analysis in different aspects of de biomedical researc

Sweet and Sour Ehrlichia: Glycoproteomics and Phosphoproteomics Reveal New Players in Ehrlichia ruminantium Physiology and Pathogenesis

Ehrlichia ruminantium; N-glycoproteins; O-GlcNAcylated proteinsEhrlichia ruminantium; N-glicoproteïnes; Proteïnes O-GlcNAciladesEhrlichia ruminantium; N-glicoproteínas; Proteínas O-GlcNAciladasUnraveling which proteins and post-translational modifications (PTMs) affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Herein, we used mass spectrometry-based assays to study protein phosphorylation and glycosylation in Ehrlichia ruminantium Gardel virulent (ERGvir) and attenuated (ERGatt) variants and, how they can modulate Ehrlichia biological processes. The characterization of the S/T/Y phosphoproteome revealed that both strains share the same set of phosphoproteins (n = 58), 36% being overexpressed in ERGvir. The percentage of tyrosine phosphorylation is high (23%) and 66% of the identified peptides are multi-phosphorylated. Glycoproteomics revealed a high percentage of glycoproteins (67% in ERGvir) with a subset of glycoproteins being specific to ERGvir (n = 64/371) and ERGatt (n = 36/343). These glycoproteins are involved in key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair, and replication. Label-free quantitative analysis revealed over-expression in 31 proteins in ERGvir and 8 in ERGatt. While further PNGase digestion confidently localized 2 and 5 N-glycoproteins in ERGvir and ERGatt, respectively, western blotting suggests that many glycoproteins are O-GlcNAcylated. Twenty-three proteins were detected in both the phospho- and glycoproteome, for the two variants. This work represents the first comprehensive assessment of PTMs on Ehrlichia biology, rising interesting questions regarding ER-host interactions. Phosphoproteome characterization demonstrates an increased versatility of ER phosphoproteins to participate in different mechanisms. The high number of glycoproteins and the lack of glycosyltransferases-coding genes highlight ER dependence on the host and/or vector cellular machinery for its own protein glycosylation. Moreover, these glycoproteins could be crucial to interact and respond to changes in ER environment. PTMs crosstalk between of O-GlcNAcylation and phosphorylation could be used as a major cellular signaling mechanism in ER. As little is known about the Ehrlichia proteins/proteome and its signaling biology, the results presented herein provide a useful resource for further hypothesis-driven exploration of Ehrlichia protein regulation by phosphorylation and glycosylation events. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD012589

Sweet and Sour Ehrlichia: Glycoproteomics and Phosphoproteomics Reveal New Players in Ehrlichia ruminantium Physiology and Pathogenesis

Unraveling which proteins and post-translational modifications (PTMs) affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Herein, we used mass spectrometry-based assays to study protein phosphorylation and glycosylation in Ehrlichia ruminantium Gardel virulent (ERGvir) and attenuated (ERGatt) variants and, how they can modulate Ehrlichia biological processes. The characterization of the S/T/Y phosphoproteome revealed that both strains share the same set of phosphoproteins (n = 58), 36% being overexpressed in ERGvir. The percentage of tyrosine phosphorylation is high (23%) and 66% of the identified peptides are multi-phosphorylated. Glycoproteomics revealed a high percentage of glycoproteins (67% in ERGvir) with a subset of glycoproteins being specific to ERGvir (n = 64/371) and ERGatt (n = 36/343). These glycoproteins are involved in key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair, and replication. Label-free quantitative analysis revealed over-expression in 31 proteins in ERGvir and 8 in ERGatt. While further PNGase digestion confidently localized 2 and 5 N-glycoproteins in ERGvir and ERGatt, respectively, western blotting suggests that many glycoproteins are O-GlcNAcylated. Twenty-three proteins were detected in both the phospho- and glycoproteome, for the two variants. This work represents the first comprehensive assessment of PTMs on Ehrlichia biology, rising interesting questions regarding ER–host interactions. Phosphoproteome characterization demonstrates an increased versatility of ER phosphoproteins to participate in different mechanisms. The high number of glycoproteins and the lack of glycosyltransferases-coding genes highlight ER dependence on the host and/or vector cellular machinery for its own protein glycosylation. Moreover, these glycoproteins could be crucial to interact and respond to changes in ER environment. PTMs crosstalk between of O-GlcNAcylation and phosphorylation could be used as a major cellular signaling mechanism in ER. As little is known about the Ehrlichia proteins/proteome and its signaling biology, the results presented herein provide a useful resource for further hypothesis-driven exploration of Ehrlichia protein regulation by phosphorylation and glycosylation events. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD012589

Aplicació de tècniques proteòmiques de quantificació i validació en recerca biomèdica

Les tècniques proteòmiques basades en l’espectrometria de masses (MS) permeten la caracterització de la complexa i dinàmica natura del proteoma. Aquestes eines han contribuit a entendre millor certes funcions biològiques i respostes cel·lulars i a obtenir biomarcadors específics per a l’estudi d’algunes malalties. En aquest treball, es van utilitzar tècniques proteòmiques de quantificació i validació, basades en l’espectrometria de masses (MS), en diferents estudis de recerca biomèdica. En el primer estudi es va comparar el perfil de proteïnes del fluid vitri de pacients diabètics amb retinopatia diabètica proliferativa (PDR) amb el de pacients no diabètics amb forat macular idiopàtic (MH). L’anàlisi proteòmica comparativa es va fer amb el sistema d’electroforesi diferencial en gel bidimensional basat en el marcatge fluorescent (2D-DIGE). Per MS es van identificar 11 proteïnes diferencials entre els pacients amb PDR i els individus no diabètics. Cinc de les proteïnes diferencials és van validar per western blot en fluids vitris i per RT-PCR en retines. L’anàlisi proteòmica 2D-DIGE va servir per a identificar potencials candidats involucrats en la patogènesis de la PDR. En el segon estudi es va estudiar l’efecte del gen REgulador AutoImmune (AIRE) mitjançant la comparació dels proteomes de cèl·lules epitelials transfectades o no amb AIRE. L’anàlisi comparativa es va realitzar combinant dues tècniques de proteòmica quantitativa: la 2D-DIGE i l’etiquetatge isotòpic codificat de proteïna (ICPL) en combinació amb cromatografia líquida acoblada a espectrometria de masses (LC-MS). Els resultats van mostrar un nivell incrementat de varies xaperones en les cèl·lules que expressaven AIRE, mentre que diferents proteïnes de interacció del citoesquelet es van trobar disminuïdes. A més, algunes proteïnes relacionades amb apoptosi tenien abundàncies diferencials entre unes cèl·lules i les altres. Aquests resultats es van confirmar per western blot i citometria de flux. Finalment, assajos d’apoptosi amb annexin V i etopòsid van demostrar que les cèl·lules positives en AIRE patien més apoptosi espontània i eren menys resistents a la inducció d’apoptosi. Els resultats obtinguts van corroborar el paper d’AIRE com a inductor d’apoptosi. En el tercer estudi, amb l’objectiu de identificar possibles proteïnes biomarcadores de l’activitat TGFβ en gliomes es van analitzar les proteïnes secretades en els cultius cel·lulars primaris derivats de tumors (PCTCs), tractats o no amb TGFβ, mitjançant experiments quantitatius de LC-MS sense marcatge i ICPL. Es van identificar varies proteïnes candidates per a les que es van desenvolupar mètodes d’anàlisi de seguiment d’una reacció seleccionada (SRM) per a poder validar-les en mostres de líquid cefaloraquidi (CSF) i plasma de pacients amb glioma. Per l’anàlisi dels CSFs els resultats van mostrar una clara correlació entre les proteïnes candidates i els nivells de TGFβ en aquestes mostres. Igualment, al analitzar les proteïnes candidates per SRM en mostres de plasma de pacients amb glioma en fases alternades de tractament amb un inhibidor de TGFβ es va observar que els nivells de les proteïnes d’interès eren modulades pel tractament. Aquestes proteïnes conformarien una firma proteica que podria ser útil per al diagnòstic i el seguiment del tractament dels pacients. En conjunt, els resultats obtinguts en cada un dels tres estudis demostren la utilitat de l’anàlisi proteòmica en diferents aspectes de la investigació biomèdica.Proteomic techniques based on mass spectrometry (MS) allow the characterization of the complex and dynamic nature of the proteome. These tools have contributed to better understand certain biological functions and cellular responses and to obtain specific biomarkers useful for the management of some diseases. In this work, quantitative and validation proteomic techniques, based on mass spectrometry, were applied to different biomedical research projects. In the first study, the protein profile of the vitreous fluid of diabetic patients with proliferative diabetic retinopathy (PDR) and non diabetic patients with macular hole (MH) was compared. Comparative proteomic analysis was performed using differential in gel electrophoresis based on fluorescent labeling (2D_DIGE). Eleven differential proteins between PDR and non diabetic patients were identified by MS. Five differential proteins were validated by western blot analysis of vitreous fluid and by RT-PCR of retinas. 2D-DIGE proteomic analysis allowed the identification of potential candidates involved in the pathogenesis of PDR. In the second study, the effect of AutoImmune Regulator (AIRE) gen was studied by comparison of the proteomic profile of epithelial cells transfected or not with AIRE. The comparative analysis was done using to proteomic techniques: 2D-DIGE and Isotopic Coded Protein Labeling (ICPL) in combination with liquid chromatography coupled to mass spectrometry (LC-MS). Results showed an increased level of some chaperons in the cells expressing AIRE, while some cytoskeleton interacting proteins were decreased. Moreover, proteins related wit apoptosis had differential abundances between samples. These results were confirmed by western blot and flow cytometry. Finally, apoptosis assays with annexin V and etoposide demonstrated that AIRE-positive cells suffer more spontaneous apoptosis and are less resistant to apoptosis induction. These results confirm the role of AIRE as an inducer of apoptosis. In the third study, the main objective was the identification of biomarker proteins of TGFβ activity in gliomas. Secreted proteins from primary cultures of tumor cells (PCTCs), treated or not with TGFβ, were analyzed by label free and ICPL LC-MS quantitative experiments. Some candidate proteins were identified. Selected reaction monitoring (SRM) methods were designed to analyze these candidate proteins in cerebrospinal fluid (CSF) and plasma of glioma patients. CSF analysis showed a clear relationship between the protein levels and the TGFβ concentration. When the SRM methods were applied to the plasma of glioma patients under alternate periods of treatment with a TGFβ inhibitor, the levels of proteins of interest were observed to be modulated by the treatment. These proteins can thus constitute a protein signature useful for diagnostic and treatment monitoring. Overall, the results obtained in each of the projects show the usefulness of the proteomic analysis in different aspects of de biomedical researc

A multicentric study to evaluate the use of relative retention times in targeted proteomics

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.All laboratories from Spain are members of ProteoRed (Plataforma de Recursos Biomoleculares y Bioinformáticos) and are supported by grant PT13/0001 funded by Instituto de Salud Carlos III (ISCIII) and FEDER.S

Inter-laboratory evaluation of instrument platforms and experimental workflows for quantitative accuracy and reproducibility assessment

The reproducibility of plasma protein quantitation between laboratories and between instrument types was examined in a large-scale international study involving 16 laboratories and 19 LC–MS/MS platforms, using two kits designed to evaluate instrument performance and one kit designed to evaluate the entire bottom-up workflow. There was little effect of instrument type on the quality of the results, demonstrating the robustness of LC/MRM-MS with isotopically labeled standards. Technician skill was a factor, as errors in sample preparation and sub-optimal LC–MS performance were evident. This highlights the importance of proper training and routine quality control before quantitation is done on patient samples