Summary of Study ST001922
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001213. The data can be accessed directly via it's Project DOI: 10.21228/M8X70P This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001922 |
| Study Title | Sublytic membrane attack complex drives glycolysis and mitochondrial dysfunction with inflammatory consequences in human monocyte-derived macrophages |
| Study Summary | The terminal stage in the complement activation pathways, the membrane attack complex (MAC), is upregulated in diabetic and rheumatoid arthritis patients, contributing pathologically by increasing inflammation. Previous research has highlighted that a sublytic dose of MAC can initiate NLRP3 inflammasome activation via calcium influx and loss of mitochondrial membrane potential. Here, we show that sublytic concentrations of MAC mediate a previously undescribed perturbation in cellular energy metabolism in human monocyte-derived macrophages, by phenotypic skewing towards glycolysis and upregulation of glycolysis-promoting genes. Sublytic MAC concentrations drive mitochondrial dysfunction, characterised by a fragmented mitochondrial morphology, loss of maximal respiratory response, depleted mitochondrial membrane potential as well as increased mitochondrial reactive oxygen species production. The consequences of these alterations in glycolytic metabolism and mitochondrial dysfunction lead to NLRP3 inflammasome activation, driving gasdermin D formation and IL-18 release. This novel link between sublytic MAC and immunometabolism, with direct consequences for downstream inflammatory processes, is important for development of novel therapeutics for areas where MAC may mediate disease. |
| Institute | GSK |
| Department | Discovery Analytical |
| Laboratory | MST-MedDesign |
| Last Name | Kozole |
| First Name | Joseph |
| Address | 1250 Collegeville Ave, Upper Providence, PA, US |
| joseph.x.kozole@gsk.com | |
| Phone | 8144410679 |
| Submit Date | 2021-09-23 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-10-18 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Combined analysis:
| Analysis ID | AN003123 | AN003124 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | Phenomenex Luna NH2 (150 x 2.1mm,3um) | Waters Acquity BEH C18 (150 x 2mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED | POSITIVE |
| Units | counts | counts |
MS:
| MS ID: | MS002904 |
| Analysis ID: | AN003123 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometric analysis was performed using data dependent acquisition. Full scan spectra were acquired at a scan range of 61 to 915 m/z at a resolution of 70,000 with an automatic gain control (AGC) of 1e6 ions and maximum injection time of 200 ms. Top 7 data dependent acquisition was employed with priority placed on a custom inclusion list built for known metabolite features. The custom inclusion list was derived from the analysis of neat standards part of the Mass Spectrometry Metabolite Library (IROA Technologies). Precursor ions were isolated with a quadrupole mass window of 2.0 m/z and collision induced dissociation fragmentation performed with stepped collision energy of 20, 30, and 45 V. MS/MS spectra were acquired at a resolution of 17,500 with an AGC target of 3e3 ions and a maximum injection time of 200 ms. Raw data was aligned, integrated, and grouped using Thermo Compound Discoverer v3.1.0.305. Deuterated L-Tryptophan, L-Phenylalanine, and Caffeine internal standards added to sample reconstitution solvents were used for data normalization. Peak annotation was based on the same database used to build the custom inclusion list for known metabolite features, and included retention time, m/z, and MS/MS data for > 500 primary metabolites. Peaks not annotated using the custom database were searched against m/z Cloud as an alternative approach to peak annotation. Statistical and pathway enrichment analysis, as well as data representation was performed using MetaboAnalyst 5.0 (Xia and Wishart 2010, Pang, Chong et al. 2021). |
| Ion Mode: | UNSPECIFIED |
| MS ID: | MS002905 |
| Analysis ID: | AN003124 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometric analysis was performed using data dependent acquisition. Full scan spectra were acquired at a scan range of 61 to 915 m/z at a resolution of 70,000 with an automatic gain control (AGC) of 1e6 ions and maximum injection time of 200 ms. Top 7 data dependent acquisition was employed with priority placed on a custom inclusion list built for known metabolite features. The custom inclusion list was derived from the analysis of neat standards part of the Mass Spectrometry Metabolite Library (IROA Technologies). Precursor ions were isolated with a quadrupole mass window of 2.0 m/z and collision induced dissociation fragmentation performed with stepped collision energy of 20, 30, and 45 V. MS/MS spectra were acquired at a resolution of 17,500 with an AGC target of 3e3 ions and a maximum injection time of 200 ms. Raw data was aligned, integrated, and grouped using Thermo Compound Discoverer v3.1.0.305. Deuterated L-Tryptophan, L-Phenylalanine, and Caffeine internal standards added to sample reconstitution solvents were used for data normalization. Peak annotation was based on the same database used to build the custom inclusion list for known metabolite features, and included retention time, m/z, and MS/MS data for > 500 primary metabolites. Peaks not annotated using the custom database were searched against m/z Cloud as an alternative approach to peak annotation. Statistical and pathway enrichment analysis, as well as data representation was performed using MetaboAnalyst 5.0 (Xia and Wishart 2010, Pang, Chong et al. 2021). |
| Ion Mode: | POSITIVE |
