Summary of Study ST002848
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 PR001783. The data can be accessed directly via it's Project DOI: 10.21228/M87M7X 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 | ST002848 |
| Study Title | Lipidomics for linked CD1 proteins |
| Study Summary | In this study, the four types of human CD1 antigen-presenting molecules were constructed using a protein linker between heavy chains of CD1 proteins and beta 2 macroglobulin (B2m), thus called linked CD1 proteins, expressed in human myelogenous cell line K562 cell, purified using affinity chromatography, and extracted using the adapted Folch method for bound lipids. These extracted lipids in triplicates from four CD1 proteins were profiled using the extraction from in parallel generated HLA-B27 protein as a control. Data demonstrated differing lipid capture motifs based on the length and chemical structures of lipids bound, pointing to general self-lipid capture mechanisms. For CD1a and CD1d, lipid size matches CD1 cleft volume, whereas CD1b shows a nearly universal size mismatch with its ligands, which results from the uniform seating of two small lipids within its large cleft. Further, the list of compounds that comprise the assembled CD1 lipidomes provides a resource for matching to bioactive lipids from other fields of research and supports the ongoing discovery of lipid blockers and antigens for T cells. |
| Institute | Brigham and Women's Hospital |
| Last Name | Huang |
| First Name | Shouxiong |
| Address | 251 Kettering Laboratory Complex, 160 Panzeca Way, Cincinnati, OH |
| shouxiong.huang@uc.edu | |
| Phone | 5135587572 |
| Submit Date | 2023-08-10 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzdata.xml |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-11-10 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001783 |
| Project DOI: | doi: 10.21228/M87M7X |
| Project Title: | Lipidomics for linked and unlinked CD1 proteins |
| Project Summary: | The cellular CD1 system binds lipid antigens for display to T cells. Here we developed a lipidomics platform that detected > 2000 distinct lipids in cellular CD1 complexes, demonstrating a broad display of self-sphingolipids and phospholipids to T cells. The four types of human CD1 antigen presenting molecules show differing lipid capture motifs based on the length and chemical structures of lipids bound, pointing to general self-lipid capture mechanisms. For CD1a and CD1d, lipid size matches CD1 cleft volume, whereas CD1b shows a nearly universal size mismatch with its ligands, which results from the uniform seating of two small lipids within its large cleft. Further, the list of compounds that comprise the assembled CD1 lipidomes provides a resource for matching to bioactive lipids from other fields of research and supports the ongoing discovery of lipid blockers and antigens for T cells. |
| Institute: | Brigham and Women's Hospital |
| Last Name: | Huang |
| First Name: | Shouxiong |
| Address: | 251 Kettering Laboratory Complex, 160 Panzeca Way, Cincinnati, OH |
| Email: | shouxiong.huang@uc.edu |
| Phone: | 5135587572 |
Subject:
| Subject ID: | SU002960 |
| 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 | Proteins bound with detected lipids |
|---|---|---|
| SA308556 | K562B27-2 | B27 control |
| SA308557 | K562B27-1 | B27 control |
| SA308558 | K562B27-3 | B27 control |
| SA308559 | Buffer-1 | Buffer control |
| SA308560 | Buffer-2 | Buffer control |
| SA308561 | Buffer-3 | Buffer control |
| SA308562 | K562CD1a-2 | CD1a |
| SA308563 | K562CD1a-1 | CD1a |
| SA308564 | K562CD1a-3 | CD1a |
| SA308565 | K562CD1b-1 | CD1b |
| SA308566 | K562CD1b-2 | CD1b |
| SA308567 | K562CD1b-3 | CD1b |
| SA308568 | K562CD1c-1 | CD1c |
| SA308569 | K562CD1c-2 | CD1c |
| SA308570 | K562CD1c-3 | CD1c |
| SA308571 | K562CD1d-1 | CD1d |
| SA308572 | K562CD1d-2 | CD1d |
| SA308573 | K562CD1d-3 | CD1d |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO002953 |
| Collection Summary: | Lipidomic profiles were generated from lipids that were extracted from K562 expressed CD1a, CD1b, CD1c, and CD1d proteins compared with peptide-binding protein HLA-B27 and buffer-only controls. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR002969 |
| Treatment Summary: | Lipidomic profiles were generated from lipids that were extracted from K562 expressed CD1a, CD1b, CD1c, and CD1d proteins compared with peptide-binding protein HLA-B27 and buffer-only controls. No other relevant treatment. |
Sample Preparation:
| Sampleprep ID: | SP002966 |
| Sampleprep Summary: | Lipid extraction. CD1 and MHC proteins were extracted in chloroform: methanol: water (2:2:1) (V: V: V) (Figure S1) at room temperature for 30 mins and centrifuged at 500g for 10 mins. The aqueous phase contained few lipids, so the combined interphase and organic phase were transferred and stored at -20°C for comparative lipidomics analysis, which was conducted in parallel with groups of lipid extracts. Mass spectrometry-based comparative lipidomics. Lipid eluents were annormalized to protein mass (20-80 μg), dried under nitrogen at 20°C, dissolved and briefly sonicated in starting mobile phase, which was 100% solvent B containing 70% hexanes, 30% isopropanol, 0.1% formic acid, and 0.05% ammonium hydroxide. Triplicate samples for each protein analyzed with blanks that were intermixed and monitored for lipid carryover, using an Agilent 1200 series HPLC autosampler with an Agilent 6520 Accurate-Mass Q-TOF MS controlled by MassHunter software. A normal phase gradient with solvent A (70% isopropanol, 30% methanol, 0.1% formic acid, and 0.05% ammonium hydroxide) and solvent B through a MonoChrom Diol column (3 μm X 150 mm X 2 mm; Varian, A0542150X020) were connected to a MetaGuard guard column (2 mm, Varian, A0542-MG2). The binary gradient was monitored with solvent B with 100% at 0-10 min, 50% at 17-22 min, 0% at 30-35 min, and 100% at 40-50 min, followed by an additional 0% for 6 min post-run for regeneration. Ionization occurred with a dual-electrospray ionization source maintained at 325°C with a drying gas flow of 5 L/min, nebulizer pressure of 30 pounds per square inch, and a capillary voltage of 5.5 kV. Positive- and negative-ion modes were typically monitored between m/z 100-3000 with the acquisition rate of 1.4 spectra/sec and 713.7 ms/spectrum. Internal calibrants (Agilent G1969-85001, m/z 121.050573, 922.009798) were continuously monitored to assess electrospray efficiency and mass accuracy. NanoESI-CID-MS was typically performed at a collision energy of 35V and an isolation width of 1.3 m/z and adjusted to optimize signal during individual experiments. |
| Sampleprep Protocol Filename: | CD1_lipidomics-090523.docx |
Combined analysis:
| Analysis ID | AN004666 |
|---|---|
| Analysis type | MS |
| Chromatography type | Normal phase |
| Chromatography system | Agilent 1200 |
| Column | First through a MetaGuard guard column (2 mm, 3 μm, Varian, A0542-MG2) and then a MonoChrom Diol column (150 X 2 mm, 3μm, Varian, A0542150X020) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6520 QTOF |
| Ion Mode | POSITIVE |
| Units | 542 |
Chromatography:
| Chromatography ID: | CH003512 |
| Chromatography Summary: | Triplicate samples for each protein analyzed with blanks that were intermixed and monitored for lipid carryover, using an Agilent 1200 series HPLC autosampler with an Agilent 6520 Accurate-Mass Q-TOF MS controlled by MassHunter software. A normal phase gradient with solvent A (70% isopropanol, 30% methanol, 0.1% formic acid, and 0.05% ammonium hydroxide) and solvent B through a MonoChrom Diol column (3 μm X 150 mm X 2 mm; Varian, A0542150X020) were connected to a MetaGuard guard column (2 mm, Varian, A0542-MG2). The binary gradient was monitored with solvent B with 100% at 0-10 min, 50% at 17-22 min, 0% at 30-35 min, and 100% at 40-50 min, followed by an additional 0% for 6 min post-run for regeneration. |
| Methods Filename: | CD1_lipidomics-090523.docx |
| Instrument Name: | Agilent 1200 |
| Column Name: | First through a MetaGuard guard column (2 mm, 3 μm, Varian, A0542-MG2) and then a MonoChrom Diol column (150 X 2 mm, 3μm, Varian, A0542150X020) |
| Column Temperature: | 22 |
| Flow Gradient: | The binary gradient was monitored with solvent B with 100% at 0-10 min, 50% at 17-22 min, 0% at 30-35 min, and 100% at 40-50 min, followed by an additional 0% for 6 min post-run for regeneration. |
| Flow Rate: | 0.7 ml/min |
| Solvent A: | 70% isopropanol, 30% methanol, 0.1% formic acid, and 0.05% ammonium hydroxide |
| Solvent B: | 70% hexanes, 30% isopropanol, 0.1% formic acid, and 0.05% ammonium hydroxide |
| Chromatography Type: | Normal phase |
MS:
| MS ID: | MS004413 |
| Analysis ID: | AN004666 |
| Instrument Name: | Agilent 6520 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Ionization occurred with a dual-electrospray ionization source maintained at 325°C with a drying gas flow of 5 L/min, nebulizer pressure of 30 pounds per square inch, and a capillary voltage of 5.5 kV. Positive- and negative-ion modes were typically monitored between m/z 100-3000 with the acquisition rate of 1.4 spectra/sec and 713.7 ms/spectrum. Internal calibrants (Agilent G1969-85001, m/z 121.050573, 922.009798) were continuously monitored to assess electrospray efficiency and mass accuracy. NanoESI-CID-MS was typically performed at a collision energy of 35V and an isolation width of 1.3 m/z and adjusted to optimize signal during individual experiments. MS data were acquired with MassHunter software. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | CD1_lipidomics-090523.docx |
