Summary of Study ST001877
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 PR001183. The data can be accessed directly via it's Project DOI: 10.21228/M8SM4R 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 | ST001877 |
| Study Title | Targeted Sphingolipid analysis of HeLa knockout for expression of GRASP55 |
| Study Summary | Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 acts by binding to these enzymes and preventing their entry to COPI derived retrograde transport vesicles thus concentrating them in the trans-Golgi. In genome edited cells lacking GRASP55 the enzymes relocate to cis-Golgi. Here we evaluated the impact of deleting GRASP55 on sphingolipid composition of HeLa cells by targeted lipid analysis. |
| Institute | IBBC, CNR |
| Last Name | parashuraman |
| First Name | seetharaman |
| Address | Via Pietro Castellino 111, Napoli, NA, 80131, Italy |
| raman@ibbc.cnr.it | |
| Phone | 0816132283 |
| Submit Date | 2021-07-18 |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-08-03 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001183 |
| Project DOI: | doi: 10.21228/M8SM4R |
| Project Title: | Targeted Sphingolipid analysis of HeLa knockout for expression of GRASP55 |
| Project Summary: | Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 acts by binding to these enzymes and preventing their entry to COPI derived retrograde transport vesicles thus concentrating them in the trans-Golgi. In genome edited cells lacking GRASP55 the enzymes relocate to cis-Golgi. Here we evaluated the impact of deleting GRASP55 on sphingolipid composition of HeLa cells by targeted lipid analysis. |
| Institute: | IBBC, CNR |
| Last Name: | parashuraman |
| First Name: | seetharaman |
| Address: | Via Pietro Castellino 111, Napoli, NA, 80131, Italy |
| Email: | raman@ibbc.cnr.it |
| Phone: | 0816132283 |
Subject:
| Subject ID: | SU001954 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA174893 | KO_3 | GRASP55 KO |
| SA174894 | KO_2 | GRASP55 KO |
| SA174895 | KO_1 | GRASP55 KO |
| SA174896 | C_2 | no treatment |
| SA174897 | C_3 | no treatment |
| SA174898 | C_1 | no treatment |
| Showing results 1 to 6 of 6 |
Collection:
| Collection ID: | CO001947 |
| Collection Summary: | Cells were washed in ice cold PBS and collected by scraping |
| Sample Type: | HeLa cells |
Treatment:
| Treatment ID: | TR001966 |
| Treatment Summary: | Cells were treated with CRISPR/Cas9 to knockout GRASP55 expression |
Sample Preparation:
| Sampleprep ID: | SP001960 |
| Sampleprep Summary: | Samples were fortified with an Internal standard Mix and lipids were extracted twice with an extraction mix consisting of 85:15 Ethyl acetate 70% Isopropanol. All lipids that were used in the Internal standard mix and in the Calibration mixes were purchased from Avanti Polar Lipids Inc. After evaporating the cell extract to dryness, the samples were reconstituted in the mobile phase. |
Combined analysis:
| Analysis ID | AN003080 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Accela 1250 |
| Column | Thermo Accucore C18 (100 x 2.1mm,2.6um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Thermo Quantum Ultra |
| Ion Mode | POSITIVE |
| Units |
Chromatography:
| Chromatography ID: | CH002252 |
| Chromatography Summary: | Samples were analyzed with a Quantum Ultra triple quadrupole mass spectrometer connected to an Accela HPLC and Accela autosampler using a solvent gradient. Ceramides identity was achieved through MRM analysis with soft fragmentation. Quantitative analysis is based on calibration curves generated for each ceramide. J. Bielawski et al. / Methods 39 (2006) 82–91 |
| Instrument Name: | Thermo Accela 1250 |
| Column Name: | Thermo Accucore C18 (100 x 2.1mm,2.6um) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002826 |
| Analysis ID: | AN003080 |
| Instrument Name: | Thermo Quantum Ultra |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Samples were analyzed with a Quantum Ultra triple quadrupole mass spectrometer connected to an Accela HPLC and Accela autosampler using a solvent gradient. Ceramides identity was achieved through MRM analysis with soft fragmentation. Quantitative analysis is based on calibration curves generated for each ceramide. J. Bielawski et al. / Methods 39 (2006) 82–91 |
| Ion Mode: | POSITIVE |
