Summary of Study ST002162
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 PR001375. The data can be accessed directly via it's Project DOI: 10.21228/M8069C This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST002162 |
| Study Title | CFAP418 participates in membrane-associated cellular processes through binding lipids during ciliogenesis |
| Study Summary | Ciliopathies and retinal degenerative diseases are heterogeneous groups of genetic diseases. CFAP418 is a causative gene of both diseases, and its sequence is evolutionarily conserved. Here, we employ affinity purification coupled with mass spectrometry and quantitative lipidomic, proteomic, and phosphoproteomic approaches to address the function of CFAP418 in retinas. We show CFAP418 unexpectedly binds to lipid metabolism precursor phosphatidic acid (PA) and mitochondrion-specific lipid cardiolipin but does not form a tight and static complex with proteins. Loss of Cfap418 leads to a widespread disruption of membrane lipid homeostasis and changes in protein-membrane association, which subsequently causes mitochondrial morphological and functional defects and membrane remodeling abnormalities in multiple vesicular trafficking pathways. The signaling of PA-binding protein kinase Ca is increased. Our results indicate that membrane lipid imbalance is a new pathological mechanism underlying inherited ciliopathies and retinal degenerations, which is associated with other known causative RAB28 and BBS genes. |
| Institute | University of Utah - Metabolomics Core |
| Last Name | Maschek |
| First Name | John |
| Address | Emma Eccles Jones Medical Science Building, 15 N Medical Dr East, Salt Lake City, UT, 84112, USA |
| alan.maschek@pharm.utah.edu | |
| Phone | 801-587-7779 |
| Submit Date | 2022-05-10 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-05-10 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001375 |
| Project DOI: | doi: 10.21228/M8069C |
| Project Title: | CFAP418 participates in membrane-associated cellular processes through binding lipids during ciliogenesis. |
| Project Summary: | Ciliopathies and retinal degenerative diseases are heterogeneous groups of genetic diseases. CFAP418 is a causative gene of both diseases, and its sequence is evolutionarily conserved. Here, we employ affinity purification coupled with mass spectrometry and quantitative lipidomic, proteomic, and phosphoproteomic approaches to address the function of CFAP418 in retinas. We show CFAP418 unexpectedly binds to lipid metabolism precursor phosphatidic acid (PA) and mitochondrion-specific lipid cardiolipin but does not form a tight and static complex with proteins. Loss of Cfap418 leads to a widespread disruption of membrane lipid homeostasis and changes in protein-membrane association, which subsequently causes mitochondrial morphological and functional defects and membrane remodeling abnormalities in multiple vesicular trafficking pathways. The signaling of PA-binding protein kinase Ca is increased. Our results indicate that membrane lipid imbalance is a new pathological mechanism underlying inherited ciliopathies and retinal degenerations, which is associated with other known causative RAB28 and BBS genes. |
| Institute: | University of Utah - Metabolomics Core |
| Last Name: | Maschek |
| First Name: | John |
| Address: | Emma Eccles Jones Medical Science Building, 15 N Medical Dr East, Salt Lake City, UT, 84112, USA |
| Email: | alan.maschek@pharm.utah.edu |
| Phone: | 801-587-7779 |
Subject:
| Subject ID: | SU002248 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Factor |
|---|---|---|
| SA207491 | 24 | Het |
| SA207492 | 22 | Het |
| SA207493 | 26 | Het |
| SA207494 | 29 | Het |
| SA207495 | 21 | Het |
| SA207496 | 30 | Het |
| SA207497 | 28 | Het |
| SA207498 | 23 | Het |
| SA207499 | 16 | Het |
| SA207500 | 15 | Het |
| SA207501 | 7 | Het |
| SA207502 | 5 | Het |
| SA207503 | 13 | Het |
| SA207504 | 2 | Het |
| SA207505 | 19 | Het |
| SA207506 | 12 | Het |
| SA207507 | 9 | KO |
| SA207508 | 8 | KO |
| SA207509 | 6 | KO |
| SA207510 | 3 | KO |
| SA207511 | 4 | KO |
| SA207512 | 10 | KO |
| SA207513 | 14 | KO |
| SA207514 | 18 | KO |
| SA207515 | 25 | KO |
| SA207516 | 1 | KO |
| SA207517 | 27 | KO |
| SA207518 | 17 | KO |
| SA207519 | 20 | KO |
| SA207520 | 37 | KO |
| SA207521 | 31 | KO |
| SA207522 | 11 | KO |
| SA207479 | blank 03 | #N/A |
| SA207480 | pooledQC 01 | #N/A |
| SA207481 | blank 02 | #N/A |
| SA207482 | blank 01 | #N/A |
| SA207483 | blank 00 | #N/A |
| SA207484 | pooledQC 02 | #N/A |
| SA207485 | pooledQC 00 | #N/A |
| SA207486 | pooledQC 06 | #N/A |
| SA207487 | pooledQC 03 | #N/A |
| SA207488 | pooledQC 05 | #N/A |
| SA207489 | pooledQC 07 | #N/A |
| SA207490 | pooledQC 04 | #N/A |
| Showing results 1 to 44 of 44 |
Collection:
| Collection ID: | CO002241 |
| Collection Summary: | Extraction of mouse retina lipids was carried out using a biphasic solvent system of cold methanol, methyl tert-butyl ether (MTBE), and PBS using the extraction method by Matyash et al. (J Lipid Res 2008, 49, (5), 1137-46). In a randomized sequence, tissue lipids (~10 mg) were extracted in bead-mill tubes (ceramic 1.4 mm, Mo-Bio, Qiagen, Germantown, MD) containing a solution of 225 mL MeOH, 750 mL MTBE, and internal standards (Lipid standard Mouse SPLASH LipidoMix at 10 mL per sample, Avanti Polar Lipids, Alabaster, AL). Samples were homogenized in one 30 second cycle and rested on ice for 1 hour with occasional vortexing. Then, 188 mL of PBS was added followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, and the upper phases were collected. Another aliquot of 750 mL MTBE was added to the bottom aqueous layer followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, the upper phases were combined and evaporated to dryness under speedvac. Lipid extracts were reconstituted in 250 mL of mobile phase B and transferred to an LC-MS vial for analysis. Concurrently, a process blank sample was prepared and then a pooled quality control (QC) sample was prepared by taking equal volumes (~50 mL) from each sample after final resuspension. |
| Sample Type: | Retina |
Treatment:
| Treatment ID: | TR002260 |
| Treatment Summary: | The retinas from 16 Cfap418 knockout and 16 heterozygous litter mates were collected at postnatal day 10. The retinas were kept at -80 degree before lipid extraction. |
Sample Preparation:
| Sampleprep ID: | SP002254 |
| Sampleprep Summary: | Extraction of mouse retina lipids was carried out using a biphasic solvent system of cold methanol, methyl tert-butyl ether (MTBE), and PBS using the extraction method by Matyash et al. (J Lipid Res 2008, 49, (5), 1137-46). In a randomized sequence, tissue lipids (~10 mg) were extracted in bead-mill tubes (ceramic 1.4 mm, Mo-Bio, Qiagen, Germantown, MD) containing a solution of 225 mL MeOH, 750 mL MTBE, and internal standards (Lipid standard Mouse SPLASH LipidoMix at 10 mL per sample, Avanti Polar Lipids, Alabaster, AL). Samples were homogenized in one 30 second cycle and rested on ice for 1 hour with occasional vortexing. Then, 188 mL of PBS was added followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, and the upper phases were collected. Another aliquot of 750 mL MTBE was added to the bottom aqueous layer followed by a brief vortex. Samples were then centrifuged at 14,000 x g for 10 minutes at 4 °C, the upper phases were combined and evaporated to dryness under speedvac. Lipid extracts were reconstituted in 250 mL of mobile phase B and transferred to an LC-MS vial for analysis. Concurrently, a process blank sample was prepared and then a pooled quality control (QC) sample was prepared by taking equal volumes (~50 mL) from each sample after final resuspension. Injection volumes of 2 uL for positive and 10 uL for negative mode, and iterative, tandem mass spectrometry was conducted using the same LC gradient at collision energies of 20 V and 27.5 V, respectively. |
Combined analysis:
| Analysis ID | AN003542 | AN003543 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Agilent 6550 | Agilent 6550 |
| Column | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Agilent 6540 QTOF | Agilent 6545 QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | pmol per sample | pmol per sample |
Chromatography:
| Chromatography ID: | CH002616 |
| Chromatography Summary: | Positive Mode RP LCMS |
| Instrument Name: | Agilent 6550 |
| Column Name: | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 65 C |
| Flow Gradient: | The chromatography gradient for both positive and negative modes started at 15% mobile phase B then increased to 30% B over 2.4 min. It sequentially increased to 48% B from 2.4 – 3.0 min, 82% B from 3 – 13.2 min, and 99% B from 13.2 – 13.8 min where it’s held until 16.7 min and returned to the initial conditions and equilibrated for 5 min. |
| Flow Rate: | 0.4 mL min |
| Solvent A: | 40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/9% acetonitrile/1% water;0.1% formic acid; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002617 |
| Chromatography Summary: | Negative Mode RP LCMS |
| Instrument Name: | Agilent 6550 |
| Column Name: | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 65 C |
| Flow Gradient: | The chromatography gradient for both positive and negative modes started at 15% mobile phase B then increased to 30% B over 2.4 min. It sequentially increased to 48% B from 2.4 – 3.0 min, 82% B from 3 – 13.2 min, and 99% B from 13.2 – 13.8 min where it’s held until 16.7 min and returned to the initial conditions and equilibrated for 5 min. |
| Flow Rate: | 0.4 mL min |
| Solvent A: | 40% water/60% acetonitrile; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/9% acetonitrile/1% water; 10 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003300 |
| Analysis ID: | AN003542 |
| Instrument Name: | Agilent 6540 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | For positive mode, the source gas temperature was set to 225 °C, with a drying gas flow of 11 L/minute, nebulizer pressure of 40 psig, sheath gas temp of 350 °C and sheath gas flow of 11 L/minute. VCap voltage is set at 3500 V, nozzle voltage 500V, fragmentor at 110 V, skimmer at 85 V and octopole RF peak at 750 V. For data processing, Agilent MassHunter (MH) Workstation and software packages MH Qualitiative and MH Quantitative were used. The pooled QC (n=8) and process blank (n=4) were injected throughout the sample queue to ensure the reliability of acquired lipidomics data. For lipid annotation, accurate mass and MS/MS matching was used with the Agilent Lipid Annotator library. Results from the positive and negative ionization modes from Lipid Annotator were merged based on the class of lipid identified. Data exported from MH Quantitative was evaluated using Excel where initial lipid targets are parsed based on the following criteria. Only lipids with relative standard deviations (RSD) less than 30% in QC samples are used for data analysis. Additionally, only lipids with background AUC counts in process blanks that are less than 30% of QC are used for data analysis. The parsed excel data tables are normalized based on the ratio to class-specific internal standards, then to sum prior to statistical analysis. |
| Ion Mode: | POSITIVE |
| MS ID: | MS003301 |
| Analysis ID: | AN003543 |
| Instrument Name: | Agilent 6545 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | For negative mode, the source gas temperature was set to 300 °C, with a drying gas flow of 11 L/minute, a nebulizer pressure of 30 psig, sheath gas temp of 350 °C and sheath gas flow 11 L/minute. VCap voltage was set at 3500 V, nozzle voltage 75 V, fragmentor at 175 V, skimmer at 75 V and octopole RF peak at 750 V. For data processing, Agilent MassHunter (MH) Workstation and software packages MH Qualitiative and MH Quantitative were used. The pooled QC (n=8) and process blank (n=4) were injected throughout the sample queue to ensure the reliability of acquired lipidomics data. For lipid annotation, accurate mass and MS/MS matching was used with the Agilent Lipid Annotator library. Results from the positive and negative ionization modes from Lipid Annotator were merged based on the class of lipid identified. Data exported from MH Quantitative was evaluated using Excel where initial lipid targets are parsed based on the following criteria. Only lipids with relative standard deviations (RSD) less than 30% in QC samples are used for data analysis. Additionally, only lipids with background AUC counts in process blanks that are less than 30% of QC are used for data analysis. The parsed excel data tables are normalized based on the ratio to class-specific internal standards, then to sum prior to statistical analysis. |
| Ion Mode: | NEGATIVE |
