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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Project:

Project ID:	PR001294
Project DOI:	doi: 10.21228/M8FQ39
Project Title:	Lyso-lipid induced oligodendrocytes maturation underlie restoration of optic nerve function
Project 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
Department:	Ophthalmology
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

Subject:

Subject ID:	SU002129
Subject Type:	Cultured cells
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Subject type: Cultured cells; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id	local_sample_id	Factor
SA192475	OLGO_PP	Olig Over
SA192476	OLGO_PP_20210319014845	Olig Over
SA192477	OLGO_PN	Olig Over
SA192478	OLGO_PN_20210318202010	Olig Over
SA192479	OLGO_NP_20210318175410	Olig Over
SA192480	OLGO_NP	Olig Over
SA192481	OLGO_NN_20210318164109	Olig Over
SA192482	Olig_NNP_3_20210317032612	Olig siRNA
SA192483	Olig_NPN_2	Olig siRNA
SA192484	Olig_NPN_2_20210317010010	Olig siRNA
SA192485	Olig_PNN_2	Olig siRNA
SA192486	Olig_NNP_3	Olig siRNA
SA192487	Olig_NNN_2	Olig siRNA
SA192488	Olig_PNN_2_20210317112047	Olig siRNA
SA192489	Olig_NNN_2_20210317100746	Olig siRNA
SA192490	Olig_NNP_2	Olig siRNA
SA192491	Olig_NNP_2_20210317074144	Olig siRNA
SA192492	Olig_PPP_2	Olig siRNA
SA192493	Olig_PPP_2_20210317043913	Olig siRNA
SA192457	OPCO_PN	OPC Over
SA192458	OPCO_PN_20210318232241	OPC Over
SA192459	OPCO_PP_20210318190709	OPC Over
SA192460	OPCO_NP_20210319003544	OPC Over
SA192461	OPCO_PP	OPC Over
SA192462	OPCO_NP	OPC Over
SA192463	OPCO_NN	OPC Over
SA192464	OPCO_NN_20210318220941	OPC Over
SA192465	OPC_PNN_1	OPC siRNA
SA192466	OPC_PNN_1_20210317134649	OPC siRNA
SA192467	OPC_NPN_1_20210317021311	OPC siRNA
SA192468	OPC_PPP_1	OPC siRNA
SA192469	OPC_NNP_1	OPC siRNA
SA192470	OPC_PPP_1_20210317055214	OPC siRNA
SA192471	OPC_NNN_1_20210317085445	OPC siRNA
SA192472	OPC_NNP_1_20210317123348	OPC siRNA
SA192473	OPC_NPN_1	OPC siRNA
SA192474	OPC_NNN_1	OPC siRNA
SA192494	RGC_over_NP	RGC Over
SA192495	RGC_over_PN	RGC Over
SA192496	repRGC_over_NN	RGC Over
SA192497	RGC_over_NN	RGC Over
SA192498	repRGC_over_PN	RGC Over
SA192499	repRGC_over_NP	RGC Over
SA192500	RGC_over_PP	RGC Over
SA192501	repRGC_over_PP	RGC Over
SA192502	RGC_si_PNN	RGC siRNA
SA192503	repRGC_si_NNN	RGC siRNA
SA192504	RGC_si_NNN	RGC siRNA
SA192505	RGC_si_NNP	RGC siRNA
SA192506	RGC_si_NPN	RGC siRNA
SA192507	repRGC_si_PPP	RGC siRNA
SA192508	repRGC_si_PNN	RGC siRNA
SA192509	repRGC_si_NNP	RGC siRNA
SA192510	repRGC_si_NPN	RGC siRNA
SA192511	RGC_si_PPP	RGC siRNA

Showing results 1 to 55 of 55

Showing results 1 to 55 of 55

Collection:

Collection ID:	CO002122
Collection Summary:	RGC cell isolation. Primary retinal ganglion cells (RGCs) were isolated according to the two-step immunopanning protocol described in a previous study (Dvoriantchikova, Degterev et al., 2014). Briefly, whole retinas of 10 postnatal day 11-12 pups were incubated in papain solution (16.5 U/mL; Worthington Biochemical, LS003127) for 30 minutes. Macrophaged and endothelial cells were removed from the cell suspension by panning with anti-macrophage antiserum (Accurate Chemical, AIA31240). RGCs were bound to the panning plates containing the antibody against Thy1.2 (VWR, 102646-060) and were then released by incubation with trypsin solution (Sigma, T9935). RGCs were plated in a plate coated with poly-d-lysine (Sigma, P6407) and Laminin (Sigma, L-6274). RGCs were either not treated (addition of RGC growth media), treated with LPC 18:0 at 10 µM in RGC growth media, or treated with LPC 18:1 at 10 µM in RGC growth media. RGCs were incubated for 24 hours and harvested the next day. OPC cell culture. Oligodendrocyte Precursor Cells (OPC) were purchased from ScienceCell Research Laboratories (R1600, Carlsbad, CA 92008) and cultured according to manufacturer’s recommendations. In brief, plates were coated with poly-d-lysine (Sigma, P6407; 2 µg/cm2) and incubated overnight at 37ºC. OPC growth media was prepared using OPC media (ScienceCell Research Laboratories, 1601) supplemented with OPC growth supplement (ScienceCell Research Laboratories, 1652) and penicillin/streptomycin solution (ScienceCell Research Laboratories, 0503). OPC differentiation medium was prepared using OPC media (ScienceCell Research Laboratories, 1601) supplemented with OPC differentiation supplement (ScienceCell Research Laboratories, 1672), fetal bovine serum (ScienceCell Research Laboratories, 0005) and penicillin/streptomycin solution (ScienceCell Research Laboratories, 0503). Poly-d-lysine coated plate was washed twice with sterile water, OPC growth media was added to each well and plate was placed in incubator to equilibrate for 15 minutes (37ºC and 5% CO2). Frozen vial containing OPCs was warmed in 37ºC water bath and gently rotated to equally suspend cells. 15,000 cells/cm2 were seeded and left overnight (~16 to ~18 hours) in incubator. Media was changed daily for 2 days. OPCs were either not treated (addition of OPC growth media), differentiated (addition of OPC differentiation media), treated with LPC 18:0 at 10 µM in OPC growth media, or treated with LPC 18:1 at 10 µM in OPC growth media. OPCs were incubated for 24 hours and harvested the next day. OPCs and oligodendrocytes were further validated using mRNA analysis following published methods (Jäkel, Agirre et al., 2019).
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR002141
Treatment Summary:	siRNA and overexpression. Cell transfection was carried using the INTERFERin transfection kit following manufacture’s recommendations (Polyplus Transfection, 89129-930). siRNA for LPCAT1, PLA2G4C, and LIPC were purchased from Ambion (Ambion, s102346, s107315, s67780 respectively), LPGAT1 and LIPC overexpression constructs were purchased from Genecopoeia (Genecopoeia, EX-mm15104-M61, EX-mm03119-M61). In brief, cells were transfected with 2 nM of siRNA or DNA in serum free cell culture media. INTERFERin reagent was added to the mix incubated at room temperature for 10 minutes and added to newly changed cell culture media for each well. Cells were incubated for 48 to 96 hours before harvesting.

Treatment ID:

TR002141

Treatment Summary:

siRNA and overexpression. Cell transfection was carried using the INTERFERin transfection kit following manufacture’s recommendations (Polyplus Transfection, 89129-930). siRNA for LPCAT1, PLA2G4C, and LIPC were purchased from Ambion (Ambion, s102346, s107315, s67780 respectively), LPGAT1 and LIPC overexpression constructs were purchased from Genecopoeia (Genecopoeia, EX-mm15104-M61, EX-mm03119-M61). In brief, cells were transfected with 2 nM of siRNA or DNA in serum free cell culture media. INTERFERin reagent was added to the mix incubated at room temperature for 10 minutes and added to newly changed cell culture media for each well. Cells were incubated for 48 to 96 hours before harvesting.

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].

Combined analysis:

Analysis ID	AN003334
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Accela 600
Column	Thermo Acclaim C30 (150 x 2.1mm,3um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	μg/ml

Chromatography:

Chromatography ID:	CH002469
Instrument Name:	Thermo Accela 600
Column Name:	Thermo Acclaim C30 (150 x 2.1mm,3um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003104
Analysis ID:	AN003334
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Lipids were identified using LipidSearch software v. 4.1.
Ion Mode:	UNSPECIFIED