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
Email	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

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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.

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.