Summary of Study ST002421
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 PR001559. The data can be accessed directly via it's Project DOI: 10.21228/M85X3W 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 | ST002421 |
| Study Title | UBXD8 lipidomics from whole cells (Part 1) |
| Study Summary | The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipidome of cells. LC-MS/MS lipidomics found significant changes in distinct lipid species in UBXD8 knockout cells, in particular in saturated or mono-unsaturated lipid species. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark in a variety of human disorders such as neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on UBXD8. |
| Institute | University of Arizona |
| Department | Immunobiology |
| Laboratory | Purdy Lab |
| Last Name | Purdy |
| First Name | John |
| Address | PO Box 245221, Tucson, Arizona, 85724, USA |
| purdylab@gmail.com | |
| Phone | 520-626-4371 |
| Submit Date | 2022-12-30 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-01-16 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001559 |
| Project DOI: | doi: 10.21228/M85X3W |
| Project Title: | UBXD8 lipidomics from whole cells |
| Project Summary: | The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipidome of cells. LC-MS/MS lipidomics found significant changes in distinct lipid species in UBXD8 knockout cells, in particular in saturated or mono-unsaturated lipid species. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark in a variety of human disorders such as neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on UBXD8. |
| Institute: | University of Arizona |
| Department: | Immunobiology |
| Laboratory: | Purdy Lab |
| Last Name: | Purdy |
| First Name: | John |
| Address: | PO Box 245221, Tucson, Arizona, 85724, USA |
| Email: | purdylab@gmail.com |
| Phone: | 520-626-4371 |
| Funding Source: | NIH R01 AI162671 |
| Contributors: | Rakesh Ganji, Joao A. Paulo, Yuecheng Xi, Ian Kline, Jiang Zhu, Christoph S. Clemen, Conrad C. Weihl, John G. Purdy, Steve P. Gygi, and Malavika Raman |
Subject:
| Subject ID: | SU002510 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | HEK293T |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Description |
|---|---|---|
| SA242347 | 20191130_pos_rep3_KO_a | UBXD8 Knockout |
| SA242348 | 20191130_pos_rep2_KO_b | UBXD8 Knockout |
| SA242349 | 20191130_neg_rep3_KO_b | UBXD8 Knockout |
| SA242350 | 20191130_pos_rep3_KO_b | UBXD8 Knockout |
| SA242351 | 20190829_neg_KO_1 | UBXD8 Knockout |
| SA242352 | 20191230_neg_rep2_KO_a | UBXD8 Knockout |
| SA242353 | 20191230_pos_rep2_KO_b | UBXD8 Knockout |
| SA242354 | 20191230_pos_rep2_KO_a | UBXD8 Knockout |
| SA242355 | 20191230_neg_rep2_KO_b | UBXD8 Knockout |
| SA242356 | 20191130_neg_rep3_KO_a | UBXD8 Knockout |
| SA242357 | 20191130_pos_rep2_KO_a | UBXD8 Knockout |
| SA242358 | 20190829_pos_KO_2 | UBXD8 Knockout |
| SA242359 | 20190829_pos_KO_1 | UBXD8 Knockout |
| SA242360 | 20191130_neg_rep2_KO_b | UBXD8 Knockout |
| SA242361 | 20191130_neg_rep2_KO_a | UBXD8 Knockout |
| SA242362 | 20190829_neg_KO_2 | UBXD8 Knockout |
| SA242363 | 20191230_neg_rep2_WT_a | Wild-type |
| SA242364 | 20191230_neg_rep2_WT_b | Wild-type |
| SA242365 | 20190829_neg_wt_2 | Wild-type |
| SA242366 | 20191230_pos_rep2_WT_b | Wild-type |
| SA242367 | 20190829_neg_wt_1 | Wild-type |
| SA242368 | 20191230_pos_rep2_WT_a | Wild-type |
| SA242369 | 20191130_pos_rep3_WT_a | Wild-type |
| SA242370 | 20191130_neg_rep3_WT_b | Wild-type |
| SA242371 | 20191130_neg_rep3_WT_a | Wild-type |
| SA242372 | 20191130_neg_rep2_WT_a | Wild-type |
| SA242373 | 20191130_pos_rep2_WT_a | Wild-type |
| SA242374 | 20191130_pos_rep2_WT_b | Wild-type |
| SA242375 | 20191130_neg_rep2_WT_b | Wild-type |
| SA242376 | 20190829_pos_wt_1 | Wild-type |
| SA242377 | 20190829_pos_wt_2 | Wild-type |
| SA242378 | 20191130_pos_rep3_WT_b | Wild-type |
| Showing results 1 to 32 of 32 |
Collection:
| Collection ID: | CO002503 |
| Collection Summary: | Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and streptomycin. Cells were maintained in a humidified, 5 % CO2 atmosphere at 37°C. The CRISPR-Cas9 gene editing system was used to generate UBXD8 knockout cell lines in HEK293T cells. Cells were grown in 6-well plates for lipidomics. Cells were washed with PBS, scraped into cold 50% methanol, centrifuged, and the cell pellets were frozen at -80˚C. Next, cells were resuspended in cold 50% methanol (1mL) and transferred to glass vials. Chloroform was added (0.5mL) and the mixture was gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration when resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. |
| Sample Type: | Epithelial cells |
Treatment:
| Treatment ID: | TR002522 |
| Treatment Summary: | Wild-type vs UBXD8 Knockout |
Sample Preparation:
| Sampleprep ID: | SP002516 |
| Sampleprep Summary: | Lipids were isolated from collected cultured cells. Cells were washed with PBS, treated with cold 50% methanol (1mL) and transferred to glass vials. Next, chloroform (0.5mL) was added and samples were gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration when resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. |
Combined analysis:
| Analysis ID | AN003942 | AN003943 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002919 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| Column Temperature: | 60 |
| Flow Gradient: | 25% to 100% |
| Flow Rate: | 0.25mL per min |
| Solvent A: | 40% water; 60% methanol 10mM ammonium formate and 0.1% formic acid |
| Solvent B: | 10% methanol; 90% isopropanol 10mM ammonium formate and 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003678 |
| Analysis ID: | AN003942 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Lipids were ionized using a heated electrospray ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra were collected using a transient time of 120 ms and an AGC setting of 1e5. Each sample was analyzed using negative and positive ion modes. The mass analyzer was calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar Lipids) were used in determining extraction efficiencies and lipid quantitation. Lipids were identified and quantified using MAVEN, and EI-MAVEN (Elucidata). UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and WT cells. Missing values in a sample were not imputed. The following lipid classes were included in the analysis: cholesteryl esters (CE), diacylglycerol (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative were followed for lipid species identification and quantification, including consideration of isotopic patterns resulting from naturally occurring 13C atoms and isomeric overlap. The following MS2 information was used to confirm each lipid species: PC fragment of 184.073 (positive mode) and tail identification using formic adduct (negative mode); PE fragment of 196.038 or the tail plus 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG fragment of 152.996 plus the identification of the FA tails (negative mode) and NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or neutral loss of 368.35 (positive). |
| Ion Mode: | POSITIVE |
| MS ID: | MS003679 |
| Analysis ID: | AN003943 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Lipids were ionized using a heated electrospray ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra were collected using a transient time of 120 ms and an AGC setting of 1e5. Each sample was analyzed using negative and positive ion modes. The mass analyzer was calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar Lipids) were used in determining extraction efficiencies and lipid quantitation. Lipids were identified and quantified using MAVEN, and EI-MAVEN (Elucidata). UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and WT cells. Missing values in a sample were not imputed. The following lipid classes were included in the analysis: cholesteryl esters (CE), diacylglycerol (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative were followed for lipid species identification and quantification, including consideration of isotopic patterns resulting from naturally occurring 13C atoms and isomeric overlap. The following MS2 information was used to confirm each lipid species: PC fragment of 184.073 (positive mode) and tail identification using formic adduct (negative mode); PE fragment of 196.038 or the tail plus 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG fragment of 152.996 plus the identification of the FA tails (negative mode) and NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or neutral loss of 368.35 (positive). |
| Ion Mode: | NEGATIVE |
