Summary of Study ST004217

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 PR002659. The data can be accessed directly via it's Project DOI: 10.21228/M8Z556 This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST004217
Study TitleLipid Alterations in ASAH1-Deficient Cells: Insights into Ceramide Accumulation and Lysosomal Dysfunction
Study SummaryLysosomes play a central role in cellular homeostasis by degrading proteins internalized through endocytosis and autophagy and recycling their components for organelle biogenesis. Lysosomal Storage Disorders (LSDs) represent a diverse group of diseases that disrupt lysosomal degradation, ion transport, and lipid metabolism. Among these, sphingolipidoses involve defects in glycosphingolipid breakdown, with gene products such as GBA1 identified, and others like SMPD1 and ASAH1 proposed, as genetic risk factors for Parkinson’s disease, although the underlying mechanisms remain poorly defined. In this dataset, we profile lipids from wildtype and ASAH1-/- HeLa cells, as well as from lysosomes isolated from these cells using LysoIP. Consistent with the loss of ASAH1 function, we observe elevated ceramide levels in knockout cells.
Institute
Harvard Medical School
Last NameHarper
First NameWade
Address25 Shattuck St, Boston, MA 02115
Emailwade_harper@hms.harvard.edu
Phone8573087183
Submit Date2025-09-19
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2025-10-14
Release Version1
Wade Harper Wade Harper
https://dx.doi.org/10.21228/M8Z556
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002659
Project DOI:doi: 10.21228/M8Z556
Project Title:Lipidomic analysis of lysosome and hela cell lipids from cells lacking ASAH1
Project Summary:Lysosomes play a central role in cellular homeostasis by degrading proteins internalized through endocytosis and autophagy and recycling their components for organelle biogenesis. Lysosomal Storage Disorders (LSDs) represent a diverse group of diseases that disrupt lysosomal degradation, ion transport, and lipid metabolism. Among these, sphingolipidoses involve defects in glycosphingolipid breakdown, with gene products such as GBA1 identified, and others like SMPD1 and ASAH1 proposed, as genetic risk factors for Parkinson’s disease, although the underlying mechanisms remain poorly defined. In this dataset, we profile lipids from wildtype and ASAH1-/- HeLa cells, as well as from lysosomes isolated from these cells using LysoIP. Consistent with the loss of ASAH1 function, we observe elevated ceramide levels in knockout cells.
Institute:Harvard Medical School
Last Name:Harper
First Name:Wade
Address:25 Shattuck St, Boston, MA 02115
Email:wade_harper@hms.harvard.edu
Phone:8573087183

Subject:

Subject ID:SU004369
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 Sample source Genotype
SA485763HeLa_WholeCell-ASAM-1_negHeLa Cells ASAM
SA485764HeLa_IP-ASAM-6_posHeLa Cells ASAM
SA485765HeLa_IP-ASAM-5_posHeLa Cells ASAM
SA485766HeLa_IP-ASAM-4_posHeLa Cells ASAM
SA485767HeLa_IP-ASAM-3_posHeLa Cells ASAM
SA485768HeLa_IP-ASAM-2_posHeLa Cells ASAM
SA485769HeLa_IP-ASAM-1_posHeLa Cells ASAM
SA485770HeLa_WholeCell-ASAM-6_negHeLa Cells ASAM
SA485771HeLa_WholeCell-ASAM-5_negHeLa Cells ASAM
SA485772HeLa_WholeCell-ASAM-3_negHeLa Cells ASAM
SA485773HeLa_WholeCell-ASAM-2_negHeLa Cells ASAM
SA485774HeLa_WholeCell-ASAM-4_negHeLa Cells ASAM
SA485775HeLa_WholeCell-ASAM-2_HeLa Cells ASAM
SA485776HeLa_WholeCell-ASAM-6_HeLa Cells ASAM
SA485777HeLa_WholeCell-ASAM-3_HeLa Cells ASAM
SA485778HeLa_WholeCell-ASAM-1_HeLa Cells ASAM
SA485779HeLa_WholeCell-ASAM-5_HeLa Cells ASAM
SA485780HeLa_WholeCell-ASAM-4_HeLa Cells ASAM
SA485781HeLa_WholeCell-CONTROL-2_negHeLa Cells Control
SA485782HeLa_WholeCell-CONTROL-3_HeLa Cells Control
SA485783HeLa_IP-CONTROL-6_posHeLa Cells Control
SA485784HeLa_IP-CONTROL-5_posHeLa Cells Control
SA485785HeLa_IP-CONTROL-4_posHeLa Cells Control
SA485786HeLa_IP-CONTROL-3_posHeLa Cells Control
SA485787HeLa_IP-CONTROL-2_posHeLa Cells Control
SA485788HeLa_IP-CONTROL-1_posHeLa Cells Control
SA485789HeLa_WholeCell-CONTROL-6_HeLa Cells Control
SA485790HeLa_WholeCell-CONTROL-5_HeLa Cells Control
SA485791HeLa_WholeCell-CONTROL-4_HeLa Cells Control
SA485792HeLa_WholeCell-CONTROL-1_HeLa Cells Control
SA485793HeLa_WholeCell-CONTROL-3_negHeLa Cells Control
SA485794HeLa_WholeCell-CONTROL-2_HeLa Cells Control
SA485795HeLa_WholeCell-CONTROL-1_negHeLa Cells Control
SA485796HeLa_WholeCell-CONTROL-6_negHeLa Cells Control
SA485797HeLa_WholeCell-CONTROL-5_negHeLa Cells Control
SA485798HeLa_WholeCell-CONTROL-4_negHeLa Cells Control
SA485799HeLa_IP-notag-2_posHeLa Cells notag
SA485800HeLa_IP-notag-6_posHeLa Cells notag
SA485801HeLa_IP-notag-5_posHeLa Cells notag
SA485802HeLa_IP-notag-4_posHeLa Cells notag
SA485803HeLa_IP-notag-3_posHeLa Cells notag
SA485804HeLa_IP-notag-1_posHeLa Cells notag
SA485805HeLa_WholeCell-notag-2_HeLa Cells notag
SA485806HeLa_WholeCell-notag-6_negHeLa Cells notag
SA485807HeLa_WholeCell-notag-3_HeLa Cells notag
SA485808HeLa_WholeCell-notag-4_negHeLa Cells notag
SA485809HeLa_WholeCell-notag-3_negHeLa Cells notag
SA485810HeLa_WholeCell-notag-2_negHeLa Cells notag
SA485811HeLa_WholeCell-notag-1_negHeLa Cells notag
SA485812HeLa_WholeCell-notag-1_HeLa Cells notag
SA485813HeLa_WholeCell-notag-6_HeLa Cells notag
SA485814HeLa_WholeCell-notag-5_HeLa Cells notag
SA485815HeLa_WholeCell-notag-4_HeLa Cells notag
SA485816HeLa_WholeCell-notag-5_negHeLa Cells notag
Showing results 1 to 54 of 54

Collection:

Collection ID:CO004362
Collection Summary:Cell Line: HeLa (human cervical cancer cells). Growth Conditions: Typically cultured in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin). Harvesting: Cells are grown to ~70–80% confluency. Washed with PBS (phosphate-buffered saline) to remove media and serum lipids. Collected by scraping or trypsinization, depending on downstream analysis. Storage: Cell pellets are flash-frozen in liquid nitrogen and stored at −80 °C until lipid extraction.
Collection Protocol Filename:HeLa sample collection for lipidomics
Sample Type:HeLa cells
Collection Duration:2hr
Storage Conditions:-80℃

Treatment:

Treatment ID:TR004378
Treatment Summary:There is no special treatment. Maintain isogenic wild-type (WT) and ASAH1-/- (acid ceramidase knockout) and no tag HeLa cell lines. Control (WT) = wildtype HeLa (used as baseline). ASAH1-/- (KO) = HeLa with ASAH1 deleted, showing ceramide accumulation. No tag = wildtype HeLa without the LysoIP tag construct (negative control for immunoprecipitation, not a biological knockout).
Cell Storage:-80 degree

Sample Preparation:

Sampleprep ID:SP004375
Sampleprep Summary:HeLa cells were cultured in 10-cm dishes and harvested at approximately 80% confluence. Cell homogenates were prepared by repeated freeze–thaw cycles, consisting of snap-freezing in liquid nitrogen followed by thawing in an ultrasonic water bath for three minutes. Lipids were extracted using the Folch method【38】. Prior to extraction, an internal SPLASH® lipidomix standard and a deuterated ganglioside standard were added for normalization. The organic phase obtained from each culture was normalized to the total soluble protein content, which was determined by the BCA Protein Assay Kit (Thermo Scientific, 23225, Waltham, MA). To improve yield and reproducibility, each sample was subjected to two sequential rounds of extraction.
Processing Storage Conditions:-20℃
Extraction Method:Folch extraction
Extract Storage:-20℃

Chromatography:

Chromatography ID:CH005326
Chromatography Summary:Lipid extracts were analyzed by high-performance liquid chromatography coupled to mass spectrometry (HPLC–MS) following a protocol adapted from【39】. Separation of the organic phase was performed on a C30 reversed-phase column (Acclaim C30, 2.1 × 250 mm, 3 μm; Thermo Fisher Scientific, Bremen, Germany) maintained at 55 °C and connected to a Vanquish Horizon UHPLC system (S/N:6516208). The UHPLC was interfaced with an Exactive Orbitrap mass spectrometer (OE240, Thermo Fisher Scientific, S/N:MM10585C) equipped with a heated electrospray ionization (HESI) probe. Dried lipid samples were reconstituted in 2:1 methanol:chloroform (v/v), and 5 μL was injected for analysis in both positive and negative ionization modes. The mobile phase A consisted of 60:40 acetonitrile:water containing 10 mM ammonium formate and 0.1% formic acid, while mobile phase B consisted of 90:10 isopropanol:acetonitrile with the same additives. Chromatographic separation was achieved with a 90-minute gradient at a flow rate of 0.2 mL/min. The gradient started at 40% B and increased to 55% over the first 7 minutes, then to 65% by 8 minutes and held until 12 minutes. Between 12 and 30 minutes, the proportion of solvent B increased to 70%, followed by a rapid increase to 88% between 30 and 31 minutes, to 95% by 51 minutes, and finally to 100% by 53 minutes, which was maintained until 73 minutes. The gradient was then reduced to 40% B within 0.1 minutes and held for 16.9 minutes for column re-equilibration. The autosampler temperature was maintained at 4 °C, and the column oven temperature was set to 55 °C throughout the run.
Instrument Name:Thermo Vanquish Horizon
Column Name:Thermo Acclaim C30 (250 x 2.1 mm, 3um)
Column Temperature:55
Flow Gradient:0.2ml/min; 0-1min: 40% B, 1-8 min: 40-55%, 8-9 min 55-65%, 9-13min:65%, 13-30min: 65-70%, 30-31min: 70- 88%, 31-51 min 88-95, 51-53min 100%, 53-75min:40%.
Flow Rate:0.2 mL/min
Solvent A:60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid
Solvent B:90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN007016
Analysis Type:MS
Chromatography ID:CH005326
Num Factors:3
Num Metabolites:749
Units:pmole/ug of protein
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