Summary of Study ST003090
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 PR001919. The data can be accessed directly via it's Project DOI: 10.21228/M8P13K 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 | ST003090 |
| Study Title | Analysis of lipid profiles of N2A-Pz1-KO cells expressing tdTOMATO-vector (mock), TMEM120A, or TMEM120B |
| Study Type | Lipidomic |
| Study Summary | N2A-Pz1-KO cells expressing tdTOMATO-vector (mock), TMEM120A, or TMEM120B were used to detect differences in levels of lipids. TMEM120A is a protein without a clear function that we previously identified to inhibit mechanically activated PIEZO2 channels. TMEM120A has structural similarity a lipid modifying enzyme (ELOVL7), and has been proposed to be necessary for adipocyte maturation & triglyceride production. We performed LC-MS/MS to determine whether TMEM120A can modify the lipid content of cells where it is expressed and utilize that data to assess whether those lipids can inhibit PIEZO2 channels. This study revealed that TMEM120A expressing cells do have larger levels of phosphatidic acid and lysophosphatidic acid, and subsequent functional studies revealed that these lipids do inhibit PIEZO2. |
| Institute | Rutgers University |
| Department | Pharmacology, Physiology and Neuroscience; Metabolomics Shared Resource |
| Laboratory | Tibor Rohacs; Xiaoyang Su |
| Last Name | Rohacs |
| First Name | Tibor |
| Address | 183 South Orange Ave, Newark, NJ, 07103 |
| rohacsti@njms.rutgers.edu | |
| Phone | 973-972-4464 |
| Submit Date | 2024-02-01 |
| Num Groups | 3 |
| Total Subjects | 9 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-07-16 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001919 |
| Project DOI: | doi: 10.21228/M8P13K |
| Project Title: | Phospholipase D regulates PIEZO2 channels via formation of phosphatidic acid |
| Project Type: | Primary research |
| Project Summary: | Mechanosensitive PIEZO2 ion channels play roles in touch, proprioception, and inflammatory pain. Currently, there are no small molecule inhibitors that selectively inhibit PIEZO2 over PIEZO1. The TMEM120A protein was shown to inhibit PIEZO2 while leaving PIEZO1 unaffected. Here we find that TMEM120A expression elevates cellular levels of phosphatidic acid and lysophosphatidic acid (LPA), aligning with its structural resemblance to lipid-modifying enzymes. Intracellular application of phosphatidic acid or LPA inhibited PIEZO2, but not PIEZO1 activity. Extended extracellular exposure to the non-hydrolyzable phosphatidic acid and LPA analogue carbocyclic phosphatidic acid (ccPA) also inhibited PIEZO2. Optogenetic activation of phospholipase D (PLD), responsible for phosphatidic acid generation, inhibited PIEZO2, but not PIEZO1. Conversely, inhibiting PLD led to increased PIEZO2 activity and increased mechanical sensitivity in mice in behavioral experiments. These findings unveil lipid regulators that selectively target PIEZO2 over PIEZO1, and identify the PLD pathway as a regulator of PIEZO2 activity. |
| Institute: | Rutgers University |
| Department: | Pharmacology, Physiology and Neuroscience; Metabolomics Shared Resource |
| Laboratory: | Tibor Rohacs; Xiaoyang Su |
| Last Name: | Rohacs |
| First Name: | Tibor |
| Address: | 183 South Orange Ave, Newark, NJ, 07103 |
| Email: | rohacsti@njms.rutgers.edu |
| Phone: | 973-972-4464 |
| Funding Source: | NIH-NINDS R01-NS055159 to T.R.; NCI-CCSG P30CA072720-5923 to the Rutgers Cancer Institute of New Jersey Metabolomics Shared Resource |
| Contributors: | Matthew Gabrielle; Yevgen Yudin; Yujue Wang; Xiaoyang Su; Tibor Rohacs |
Subject:
| Subject ID: | SU003205 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Transfection |
|---|---|---|---|
| SA332976 | Mock_1 | Neuro 2A cells | Mock |
| SA332977 | Mock_3 | Neuro 2A cells | Mock |
| SA332978 | Mock_2 | Neuro 2A cells | Mock |
| SA332979 | TMEM120A_3 | Neuro 2A cells | TMEM120A |
| SA332980 | TMEM120A_2 | Neuro 2A cells | TMEM120A |
| SA332981 | TMEM120A_1 | Neuro 2A cells | TMEM120A |
| SA332982 | TMEM120B_3 | Neuro 2A cells | TMEM120B |
| SA332983 | TMEM120B_2 | Neuro 2A cells | TMEM120B |
| SA332984 | TMEM120B_1 | Neuro 2A cells | TMEM120B |
| Showing results 1 to 9 of 9 |
Collection:
| Collection ID: | CO003198 |
| Collection Summary: | N2A-Pz1-KO cells were used. One million cells per group were centrifuged, and washed with ice-cold PBS. Each cell pellet was resuspended in 500 µL of resuspension buffer (0.05 M HCl, 49% Methanol, 1% SPLASH Lipidomic Mass Spec Standard), and 1 mL methyl-tert-butyl-ether was added. Samples were vortexed for 30 seconds, and 600 µL of the organic layer was transferred to a new tube. Samples were allowed to dry overnight and stored at -80°C. |
| Sample Type: | Neuro2A cells |
| Volumeoramount Collected: | 600 µL of organic phase of lipid extraction (see Sample Prep Information) |
| Collection Vials: | 1.5 mL Eppendorf tubes |
| Storage Vials: | 1.5 mL Eppendorf tubes |
| Collection Tube Temp: | Room temp |
| Additives: | Aventi SPLASH Lipidomic Mass Spec Standard |
Treatment:
| Treatment ID: | TR003214 |
| Treatment Summary: | N2A-Pz1-KO cells were transfected using Polyethylenimine Max with 1500 ng of the vectors for the expression of tdTOMATO (mock), TMEM120A, or TMEM120B. 48 hours after transfection, the cells were treated with trypsin for 3 minutes at 37°C for collection, and counted via hemocytometer. One million cells per group were centrifuged to remove media and trypsin, and then washed with ice-cold PBS before the lipid extraction was performed. |
Sample Preparation:
| Sampleprep ID: | SP003211 |
| Sampleprep Summary: | N2A-Pz1-KO cells were transfected using PEI-Max (Polysciences Inc) with 1500 ng ptdTomato-N1 vector, 1500 ng TMEM120A-Tom, or 1500 ng TMEM120B. Cells were harvested 48 hours after transfection. One million cells per group were centrifuged, and washed with ice-cold PBS. Each cell pellet was resuspended in 500 µL of resuspension buffer (0.05 M HCl, 49% Methanol, 1% SPLASH Lipidomic Mass Spec Standard), and 1 mL methyl-tert-butyl-ether was added. Samples were vortexed for 30 seconds, and 600 µL of the organic layer was transferred to a new tube. Samples were allowed to dry overnight and stored at -80°C. |
Combined analysis:
| Analysis ID | AN005054 | AN005055 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish Horizon | Thermo Vanquish Horizon |
| Column | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) |
| 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 | Ion counts | Ion counts |
Chromatography:
| Chromatography ID: | CH003818 |
| Chromatography Summary: | Lipidomics method (Vanquish Horizon) |
| Instrument Name: | Thermo Vanquish Horizon |
| Column Name: | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) |
| Column Temperature: | 55C |
| Flow Gradient: | 0 min, 25% B; 2 min, 25% B; 5.5 min, 65% B; 12.5 min, 100% B; 19.5 min, 100% B; 20.0 min, 25% B; 30 min, 25% B |
| Flow Rate: | 0.2mL/min |
| Solvent A: | 90% water/10% methanol; 34.2 mM acetic acid; 1 mM ammonium acetate |
| Solvent B: | 75% isopropanol/25% methanol; 34.2 mM acetic acid; 1 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004792 |
| Analysis ID: | AN005054 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Positive Ionization mode with ddMS2 |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 300 |
| Ion Source Temperature: | 360 |
| Spray Voltage: | 3500 |
| Automatic Gain Control: | 3000000 |
| MS ID: | MS004793 |
| Analysis ID: | AN005055 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Negative Ionization mode with ddMS2 |
| Ion Mode: | NEGATIVE |
