Identification of components and metabolites of traditional Chinese medicines(TCMs)employing liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF MS)techniques with information-dependent acquisit...Identification of components and metabolites of traditional Chinese medicines(TCMs)employing liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF MS)techniques with information-dependent acquisition(IDA)approaches is increasingly frequent.A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings.Sequential window acquisition of all theoretical fragment-ion spectra(SWATH)is a dataindependent acquisition(DIA)method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion.Herein,the superiority of SWATH on the detection of TCMs’components was firstly investigated by comparing the detection efficiency of SWATH-MS and IDA-MS data acquisition modes,and sanguisorbin extract was used as a mode TCM.After optimizing the setting parameters of SWATH,rolling collision energy(CE)and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window.More importantly,the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition.In IDA mode,18 kinds of sanguisorbins were detected in sanguisorbin extract.A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes.Besides,26 metabolites of sanguisorbins were identified in rat plasma,and their metabolic pathways could be deduced as decarbonylation,oxidization,reduction,methylation,and glucuronidation according to their fragmental ions acquired in SWATH-MS mode.Thus,SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.展开更多
The sensorimotor and histological aspects of peripheral neuropathies were already studied by our team in two rat models:the sciatic nerve crush and the Charcot-Marie-Tooth-1A disease.In this study,we sought to highlig...The sensorimotor and histological aspects of peripheral neuropathies were already studied by our team in two rat models:the sciatic nerve crush and the Charcot-Marie-Tooth-1A disease.In this study,we sought to highlight and compare the protein signature of these two pathological situations.Indeed,the identification of protein profiles in diseases can play an important role in the development of pharmacological targets.In fact,Charcot-Marie-Tooth-1A rats develop motor impairments that are more severe in the hind limbs.Therefore,for the first time,protein expression in sciatic nerve of Charcot-Marie-Tooth-1A rats was examined.First,distal sciatic nerves were collected from Charcot-Marie-Tooth-1A and uninjured wild-type rats aged 3 months.After protein extraction,sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry was employed.445 proteins mapped to Swiss-Prot or trEMBL Uniprot databases were identified and quantified.Of these,153 proteins showed statistically significant differences between Charcot-Marie-Tooth-1A and wild-type groups.The majority of these proteins were overexpressed in Charcot-Marie-Tooth-1A.Hierarchical clustering and functional enrichment using Gene Ontology were used to group these proteins based on their biological effects concerning Charcot-Marie-Tooth-1A pathophysiology.Second,proteomic characterization of wild-type rats subjected to sciatic nerve crush was performed sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry.One month after injury,distal sciatic nerves were collected and analyzed as described above.Out of 459 identified proteins,92 showed significant differences between sciatic nerve crush and the uninjured wild-type rats used in the first study.The results suggest that young adult Charcot-Marie-Tooth-1A rats(3 months old)develop compensatory mechanisms at the level of redox balance,protein folding,myelination,and axonogenesis.These mechanisms seem insufficient to hurdle the progress of the disease.Notably,response to oxidative stress appears to be a significant feature of Charcot-Marie-Tooth-1A,potentially playing a role in the pathological process.In contrast to the first experiment,the majority of the proteins that differed from wild-type were downregulated in the sciatic nerve crush group.Functional enrichment suggested that neurogenesis,response to axon injury,and oxidative stress were important biological processes.Protein analysis revealed an imperfect repair at this time point after injury and identified several distinguishable proteins.In conclusion,we suggest that peripheral neuropathies,whether of a genetic or traumatic cause,share some common pathological pathways.This study may provide directions for better characterization of these models and/or identifying new specific therapeutic targets.展开更多
There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways.By employing the SWATH-MS technique,we quantified absolute amounts of up to thousands of protei...There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways.By employing the SWATH-MS technique,we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes.Combining SWATH-MS-based network modeling and experimental validation,we found that when RIP1 level is below~1000 molecules/cell(mpc),the cell solely undergoes TRADD-dependent apoptosis.When RIP1 is above~1000 mpc,pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount,which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical obser-vations that RIP1 is required for necroptosis but its increase down-regulates necroptosis.Higher amount of RIP1(>~46,000 mpc)suppresses apoptosis,leading to necroptosis alone.The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic.Our study provides a resource for encoding the com-plexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes,enabling RIP1 to play diverse roles in governing cell fate decisions.展开更多
Background The identification of circulating biomarkers that closely correlate with Parkinson’s Disease(PD)has failed several times in the past.Nevertheless,in this pilot study,a translational approach was conducted,...Background The identification of circulating biomarkers that closely correlate with Parkinson’s Disease(PD)has failed several times in the past.Nevertheless,in this pilot study,a translational approach was conducted,allowing the evaluation of the plasma levels of two mitochondrial-related proteins,whose combination leads to a robust model with potential diagnostic value to discriminate the PD patients from matched controls.Methods The proposed translational approach was initiated by the analysis of secretomes from cells cultured under control or well-defined oxidative stress conditions,followed by the identification of proteins related to PD pathologic mechanisms that were altered between the two states.This pipeline was further translated into the analysis of undepleted plasma samples from 28 control and 31 PD patients.Results From the secretome analysis,several mitochondria-related proteins were found to be differentially released between control and stress conditions and to be able to distinguish the two secretomes.Similarly,two mitochondrial-related proteins were found to be significantly changed in a PD cohort compared to matched controls.Moreover,a linear discriminant model with potential diagnostic value to discriminate PD patients was obtained using the combination of these two proteins.Both proteins are associated with apoptotic mitochondrial changes,which may correspond to potential indicators of cell death.Moreover,one of these proteins,the VPS35 protein,was reported in plasma for the first time,and its quantification was only possible due to its previous identification in the secretome analysis.Conclusions In this work,an adaptation of a translational pipeline for biomarker selection was presented and transposed to neurological diseases,in the present case Parkinson’s Disease.The novelty and success of this pilot study may arise from the combination of:i)a translational research pipeline,where plasma samples are interrogated using knowledge previously obtained from the evaluation of cells’secretome under oxidative stress;ii)the combined used of statistical analysis and an informed selection of candidates based on their link with relevant disease mechanisms,and iii)the use of SWATH-MS,an untargeted MS method that allows a complete record of the analyzed samples and a targeted data extraction of the quantitative values of proteins previously identified.展开更多
基金the National Natural Science Foundation of China(81573559,81530098)the Ministry of National Science and Technique(Grant No.2017ZX09309027)。
文摘Identification of components and metabolites of traditional Chinese medicines(TCMs)employing liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF MS)techniques with information-dependent acquisition(IDA)approaches is increasingly frequent.A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings.Sequential window acquisition of all theoretical fragment-ion spectra(SWATH)is a dataindependent acquisition(DIA)method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion.Herein,the superiority of SWATH on the detection of TCMs’components was firstly investigated by comparing the detection efficiency of SWATH-MS and IDA-MS data acquisition modes,and sanguisorbin extract was used as a mode TCM.After optimizing the setting parameters of SWATH,rolling collision energy(CE)and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window.More importantly,the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition.In IDA mode,18 kinds of sanguisorbins were detected in sanguisorbin extract.A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes.Besides,26 metabolites of sanguisorbins were identified in rat plasma,and their metabolic pathways could be deduced as decarbonylation,oxidization,reduction,methylation,and glucuronidation according to their fragmental ions acquired in SWATH-MS mode.Thus,SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.
基金supported by a doctoral fellowship from the European Union(European Regional Development Fund).
文摘The sensorimotor and histological aspects of peripheral neuropathies were already studied by our team in two rat models:the sciatic nerve crush and the Charcot-Marie-Tooth-1A disease.In this study,we sought to highlight and compare the protein signature of these two pathological situations.Indeed,the identification of protein profiles in diseases can play an important role in the development of pharmacological targets.In fact,Charcot-Marie-Tooth-1A rats develop motor impairments that are more severe in the hind limbs.Therefore,for the first time,protein expression in sciatic nerve of Charcot-Marie-Tooth-1A rats was examined.First,distal sciatic nerves were collected from Charcot-Marie-Tooth-1A and uninjured wild-type rats aged 3 months.After protein extraction,sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry was employed.445 proteins mapped to Swiss-Prot or trEMBL Uniprot databases were identified and quantified.Of these,153 proteins showed statistically significant differences between Charcot-Marie-Tooth-1A and wild-type groups.The majority of these proteins were overexpressed in Charcot-Marie-Tooth-1A.Hierarchical clustering and functional enrichment using Gene Ontology were used to group these proteins based on their biological effects concerning Charcot-Marie-Tooth-1A pathophysiology.Second,proteomic characterization of wild-type rats subjected to sciatic nerve crush was performed sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry.One month after injury,distal sciatic nerves were collected and analyzed as described above.Out of 459 identified proteins,92 showed significant differences between sciatic nerve crush and the uninjured wild-type rats used in the first study.The results suggest that young adult Charcot-Marie-Tooth-1A rats(3 months old)develop compensatory mechanisms at the level of redox balance,protein folding,myelination,and axonogenesis.These mechanisms seem insufficient to hurdle the progress of the disease.Notably,response to oxidative stress appears to be a significant feature of Charcot-Marie-Tooth-1A,potentially playing a role in the pathological process.In contrast to the first experiment,the majority of the proteins that differed from wild-type were downregulated in the sciatic nerve crush group.Functional enrichment suggested that neurogenesis,response to axon injury,and oxidative stress were important biological processes.Protein analysis revealed an imperfect repair at this time point after injury and identified several distinguishable proteins.In conclusion,we suggest that peripheral neuropathies,whether of a genetic or traumatic cause,share some common pathological pathways.This study may provide directions for better characterization of these models and/or identifying new specific therapeutic targets.
基金the National Natural Science Foundation of China(Grants No.81788101,11675134,11704318,81630042 and 31420103910)the China Postdoctoral Science Foundation(Grant No.2016M602071)the 111 Project(Grant Nos.B16029 and B12001).
文摘There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways.By employing the SWATH-MS technique,we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes.Combining SWATH-MS-based network modeling and experimental validation,we found that when RIP1 level is below~1000 molecules/cell(mpc),the cell solely undergoes TRADD-dependent apoptosis.When RIP1 is above~1000 mpc,pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount,which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical obser-vations that RIP1 is required for necroptosis but its increase down-regulates necroptosis.Higher amount of RIP1(>~46,000 mpc)suppresses apoptosis,leading to necroptosis alone.The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic.Our study provides a resource for encoding the com-plexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes,enabling RIP1 to play diverse roles in governing cell fate decisions.
基金This work was financed by the European Regional Development Fund(ERDF)through the COMPETE 2020-Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT–Fundação para a Ciência e a Tecnologia,I.P.,under projects:POCI-01-0145-FEDER-029311(ref.:PTDC/BTM-TEC/29311/2017),PTDC/NEU-NMC/0205/2012,UIDB/04539/2020,POCI-01-0145-FEDER-016428(ref.:SAICTPAC/0010/2015),POCI-01-0145-FEDER-016795(ref.:PTDC/NEU-SCC/7051/2014),and POCI-01-0145-FEDER-029516(PTDC/MED-NEU/29516/2017).
文摘Background The identification of circulating biomarkers that closely correlate with Parkinson’s Disease(PD)has failed several times in the past.Nevertheless,in this pilot study,a translational approach was conducted,allowing the evaluation of the plasma levels of two mitochondrial-related proteins,whose combination leads to a robust model with potential diagnostic value to discriminate the PD patients from matched controls.Methods The proposed translational approach was initiated by the analysis of secretomes from cells cultured under control or well-defined oxidative stress conditions,followed by the identification of proteins related to PD pathologic mechanisms that were altered between the two states.This pipeline was further translated into the analysis of undepleted plasma samples from 28 control and 31 PD patients.Results From the secretome analysis,several mitochondria-related proteins were found to be differentially released between control and stress conditions and to be able to distinguish the two secretomes.Similarly,two mitochondrial-related proteins were found to be significantly changed in a PD cohort compared to matched controls.Moreover,a linear discriminant model with potential diagnostic value to discriminate PD patients was obtained using the combination of these two proteins.Both proteins are associated with apoptotic mitochondrial changes,which may correspond to potential indicators of cell death.Moreover,one of these proteins,the VPS35 protein,was reported in plasma for the first time,and its quantification was only possible due to its previous identification in the secretome analysis.Conclusions In this work,an adaptation of a translational pipeline for biomarker selection was presented and transposed to neurological diseases,in the present case Parkinson’s Disease.The novelty and success of this pilot study may arise from the combination of:i)a translational research pipeline,where plasma samples are interrogated using knowledge previously obtained from the evaluation of cells’secretome under oxidative stress;ii)the combined used of statistical analysis and an informed selection of candidates based on their link with relevant disease mechanisms,and iii)the use of SWATH-MS,an untargeted MS method that allows a complete record of the analyzed samples and a targeted data extraction of the quantitative values of proteins previously identified.
