Summary of Study ST002047

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 PR001294. The data can be accessed directly via it's Project DOI: 10.21228/M8FQ39 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	ST002047
Study Title	Lyso-lipid induced oligodendrocytes maturation underlie restoration of optic nerve function
Study Summary	Protein hyper-deimination and deficiency of lyso-phospholipids (LPC 18:1) has been associated with the pathology of demyelinating disease in both humans and mice. We uncovered interesting biology of LPC 18:1, in which LPC 18:1 induced optic nerve function restoration through oligodendrocyte maturation and remyelination in mouse model systems. Our in vitro studies show LPC 18:1 protection against neuron-ectopic hyper-deimination and stimulation of oligodendrocyte maturation, while in vivo investigations recorded optic nerve function improvement following optic nerve injections of LPC 18:1, in contrast to LPC 18:0. Thus just a change in a single bond renders a dramatic alternation in biological function. The incorporation of isobaric C13-histidine in newly synthesized myelin proteins and quantitative proteome shifts are consistent with remyelination underlying restoration in optic nerve function. These results suggest that exogenous LPC 18:1 may provide a therapeutic avenue for stemming vision loss in demyelinating diseases.
Institute	University of Miami
Last Name	Bhattacharya
First Name	Sanjoy K.
Address	1638 NW 10th Avenue, Room 706-A, Miami, FL 33136
Email	sbhattacharya@med.miami.edu
Phone	3054824103
Submit Date	2021-12-16
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2022-01-21
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP002135
Sampleprep Summary:	15 µg of total protein from homogenate sample were added to 4 times the volume of acetone at 20°C and incubated at 20°C overnight. Samples were centrifuged at 21,000 g at 4°C for 30 minutes (Thermo Fisher Megafuge 8R). Supernatant was discarded and pellet (very small) was air dried for 10 minutes. The pellet was re-suspended, denatured and reduced with 6 M urea, 10 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature. Following denaturing and reduction, the re-suspended pellet was alkylated with 15 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 minutes while maintained in darkness. Alkylation reaction was quench using 20 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature while kept in darkness. Sample was diluted using 50 mM ammonium bicarbonate to contain 1 M urea. Sample was digested using either trypsin or chymotrypsin (Promega, V5111, V106A) at a 1:30 (w/w) ratio of enzyme to protein. Digestion was incubated overnight at 37°C. Reaction was terminated using 50% formic acid at a 5:100 (v/v) ratio of formic acid to sample volume. Samples were stored at -20°C or immediately desalted. Samples were desalted using the Pierce Graphite Spin Columns (Thermo, 88302) following manufacturer’s recommendations. Samples were then evaporated using the CentriVap Concentrator system (Labconco, Kansas City, MO 64132-2696) and resuspended in 30 µl of protein resuspension solution [2% (v/v) acetonitrile, 0.1% (v/v) formic acid in mass spectrometry grade water]. Lipids were harvested by adding 400 µl of 1:1 v/v methanol/chloroform mix to 200 µl of cell culture lysate. Lysate was vortexed and incubated in ice for 5 minutes, followed by the addition of 350 µl of chloroform. Lysate was centrifuged at 18,000g for 15 minutes at 4ºC. The organic layer containing lipids (bottom layer) was transferred to a new tube and desiccated using the CentriVap Concentrator system. Lipids were resuspended in 50 µl of lipid resuspension solution [50% v/v isopropyl alcohol and 50% v/v acetonitrile].

Sampleprep ID:

SP002135

Sampleprep Summary:

15 µg of total protein from homogenate sample were added to 4 times the volume of acetone at 20°C and incubated at 20°C overnight. Samples were centrifuged at 21,000 g at 4°C for 30 minutes (Thermo Fisher Megafuge 8R). Supernatant was discarded and pellet (very small) was air dried for 10 minutes. The pellet was re-suspended, denatured and reduced with 6 M urea, 10 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature. Following denaturing and reduction, the re-suspended pellet was alkylated with 15 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 minutes while maintained in darkness. Alkylation reaction was quench using 20 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature while kept in darkness. Sample was diluted using 50 mM ammonium bicarbonate to contain 1 M urea. Sample was digested using either trypsin or chymotrypsin (Promega, V5111, V106A) at a 1:30 (w/w) ratio of enzyme to protein. Digestion was incubated overnight at 37°C. Reaction was terminated using 50% formic acid at a 5:100 (v/v) ratio of formic acid to sample volume. Samples were stored at -20°C or immediately desalted. Samples were desalted using the Pierce Graphite Spin Columns (Thermo, 88302) following manufacturer’s recommendations. Samples were then evaporated using the CentriVap Concentrator system (Labconco, Kansas City, MO 64132-2696) and resuspended in 30 µl of protein resuspension solution [2% (v/v) acetonitrile, 0.1% (v/v) formic acid in mass spectrometry grade water]. Lipids were harvested by adding 400 µl of 1:1 v/v methanol/chloroform mix to 200 µl of cell culture lysate. Lysate was vortexed and incubated in ice for 5 minutes, followed by the addition of 350 µl of chloroform. Lysate was centrifuged at 18,000g for 15 minutes at 4ºC. The organic layer containing lipids (bottom layer) was transferred to a new tube and desiccated using the CentriVap Concentrator system. Lipids were resuspended in 50 µl of lipid resuspension solution [50% v/v isopropyl alcohol and 50% v/v acetonitrile].