Summary of Study ST001674

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 PR001077. The data can be accessed directly via it's Project DOI: 10.21228/M8GT3N This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001674
Study Title	Untargeted phospholipid analysis of HeLa silenced for or overexpressing GOLPH3
Study Summary	A group of sequentially-acting enzymes operating at the branchpoint among sphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternae inter-conversion mechanisms. Through these effects, GOLPH3 controls the sub-Golgi localisation, and the lysosomal degradation rate of specific enzymes. Here we evaluated the impact of overexpressing or silencing GOLPH3 on the glycerophospholipid composition of HeLa cells by untargeted lipid analysis.
Institute	École polytechnique fédérale de Lausanne (EPFL)
Department	IBI
Laboratory	UPDANGELO
Last Name	D'Angelo
First Name	Giovanni
Address	Station 15, Lausanne, Vaud, 1015, Switzerland
Email	giovanni.dangelo@epfl.ch
Phone	+41 216934276
Submit Date	2021-01-29
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2021-02-26
Release Version	1

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Project:

Project ID:	PR001077
Project DOI:	doi: 10.21228/M8GT3N
Project Title:	Untargeted phospholipid analysis of HeLa silenced for or overexpressing GOLPH3
Project Summary:	A group of sequentially-acting enzymes operating at the branchpoint among sphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternae inter-conversion mechanisms. Through these effects, GOLPH3 controls the sub-Golgi localisation, and the lysosomal degradation rate of specific enzymes. Here we evaluated the impact of overexpressing or silencing GOLPH3 on the glycerophospholipid composition of HeLa cells by untargeted lipid analysis.
Institute:	École polytechnique fédérale de Lausanne (EPFL)
Department:	IBI
Laboratory:	UPDANGELO
Last Name:	D'Angelo
First Name:	Giovanni
Address:	Station 15, Lausanne, Vaud, 1015, Switzerland
Email:	giovanni.dangelo@epfl.ch
Phone:	+41 216934276

Subject:

Subject ID:	SU001751
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
SA154479	GOLPH3_OE_1	GOLPH3 overexpression
SA154480	GOLPH3_OE_2	GOLPH3 overexpression
SA154481	GOLPH3_KD_2	GOLPH3 silencing
SA154482	GOLPH3_KD_1	GOLPH3 silencing
SA154483	CTRL_2	no treatment
SA154484	CTRL_1	no treatment

Showing results 1 to 6 of 6

Showing results 1 to 6 of 6

Collection:

Collection ID:	CO001744
Collection Summary:	Cells were collected by scraping after ice cold PBS washes
Sample Type:	HeLa cells

Treatment:

Treatment ID:	TR001764
Treatment Summary:	HeLa cells were treated to overexpress or down regulate the protein GOLPH3

Sample Preparation:

Sampleprep ID:	SP001757
Sampleprep Summary:	Total lipid extracts were prepared using a standard MTBE protocol followed by a methylamine treatment for total lipid analysis by mass spectrometry. Briefly, cell pellets or viral fractions were resuspended in 100 μL H2O. 360 μL methanol and 1.2 mL of MTBE were added and samples were placed for 10 min on a vortex at 4 C followed by incubation for 1 h at room temperature on a shaker. Phase separation was induced by addition of 200 μL of H2O. After 10 min at room temperature, samples were centrifuged at 1000 g for 10 min. The upper (organic) phase was transferred into a glass tube and the lower phase was re-extracted with 400 μL artificial upper phase [MTBE/methanol/H2O (10:3:1.5, v/v/v)]. The combined organic phases were dried in a vacuum concentrator. Lipids where then resuspended in 500 μL of ChCl3.

Sampleprep ID:

SP001757

Sampleprep Summary:

Total lipid extracts were prepared using a standard MTBE protocol followed by a methylamine treatment for total lipid analysis by mass spectrometry. Briefly, cell pellets or viral fractions were resuspended in 100 μL H2O. 360 μL methanol and 1.2 mL of MTBE were added and samples were placed for 10 min on a vortex at 4 C followed by incubation for 1 h at room temperature on a shaker. Phase separation was induced by addition of 200 μL of H2O. After 10 min at room temperature, samples were centrifuged at 1000 g for 10 min. The upper (organic) phase was transferred into a glass tube and the lower phase was re-extracted with 400 μL artificial upper phase [MTBE/methanol/H2O (10:3:1.5, v/v/v)]. The combined organic phases were dried in a vacuum concentrator. Lipids where then resuspended in 500 μL of ChCl3.

Combined analysis:

Analysis ID	AN002732
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Shimadzu Prominence UFPLC xr system
Column	HILIC Kinetex (50 x 2.1mm,2.6um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap
Ion Mode	POSITIVE
Units	intensities

Chromatography:

Chromatography ID:	CH002018
Chromatography Summary:	Lipid extracts (2 μL injection volume in ChCl3:MeOH 2:1) were separated over an 8 minutes gradient at a flow rate of 200 μL/min on a HILIC Kinetex Column (2.6lm, 2.1 × 50 mm2) on a Shimadzu Prominence UFPLC xr system (Tokyo, Japan). Mobile phase A was acetonitrile:methanol 10:1 (v/v) containing 10 mM ammonium formate and 0.5% formic acid while mobile phase B was deionized water containing 10 mM ammonium formate and 0.5% formic acid. The elution of the gradient began with 5% B at a 200 μL/min flow and increased linearly to 50% B over 7 min, then the elution continued at 50% B for 1.5 min and finally, the column was re-equilibrated for 2.5 min.
Instrument Name:	Shimadzu Prominence UFPLC xr system
Column Name:	HILIC Kinetex (50 x 2.1mm,2.6um)
Flow Gradient:	The elution of the gradient began with 5% B and increased linearly to 50% B over 7 min, then the elution continued at 50% B for 1.5 min and finally, the column was re-equilibrated for 2.5 min.
Flow Rate:	200 µL/min
Solvent A:	91% acetonitrile/9% methanol; 0.5% formic acid; 10 mM ammonium formate
Solvent B:	1005 water; 0.5% formic acid; 10 mM ammonium formate
Chromatography Type:	HILIC

MS:

MS ID:	MS002529
Analysis ID:	AN002732
Instrument Name:	Thermo Orbitrap Elite Hybrid Ion Trap-Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS data were acquired in full-scan mode at high resolution on a hybrid Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany). The system was operated at 240,000 resolution (m/z 400) with an AGC set at 1.0E6 and one microscan set at 10-ms maximum injection time. The heated electrospray source HESI II was operated in positive mode at a temperature of 90 C and a source voltage at 4.0KV. Sheath gas and auxiliary gas were set at 20 and 5 arbitrary units, respectively, while the transfer capillary temperature was set to 275 °C. Mass spectrometry data were acquired with LTQ Tuneplus2.7SP2 and treated with Xcalibur 4.0QF2 (Thermo Fisher Scientific). Lipid identification was carried out with Lipid Data Analyzer II (LDA v. 2.6.3, IGB-TUG Graz University) (Hartler et al., 2011). The LDA algorithm identifies peaks by their respective retention time, m/z and intensity. Care was taken to calibrate the instrument regularly to ensure a mass accuracy consistently lower than 3 ppm thereby leaving only few theoretical possibilities for elemental assignment.
Ion Mode:	POSITIVE