Summary of Study ST004058

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 PR002548. The data can be accessed directly via it's Project DOI: 10.21228/M8926N 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	ST004058
Study Title	Targeted Lipid and Metabolite Profiling in ATP13A2 knockout (KO) in HAP1 cells
Study Summary	Targeted profiling of lipids and metabolites was performed in ATP13A2 knockout (KO) HAP1 cells with and without polyamine (Spermine) and lipid (DOPC or PG 22:6) treatments.
Institute	Denali Therapeutics
Last Name	Suh
First Name	Jung
Address	161 Oyster Point Blvd
Email	suh@dnli.com
Phone	+1 6507973837
Submit Date	2025-06-23
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-07-23
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002548
Project DOI:	doi: 10.21228/M8926N
Project Title:	Lysosomal polyamine storage upon ATP13A2 loss impairs β-glucocerebrosidase via altered lysosomal pH and electrostatic hydrolase-lipid interactions
Project Type:	Preclinical Mouse and cellular studies
Project Summary:	ATP13A2 is an endolysosomal polyamine transporter mutated in several neurodegenerative conditions involving lysosomal defects, including Parkinson’s disease (PD). While polyamines are polybasic and polycationic molecules that play pleiotropic cellular roles, their specific impact on lysosomal health is unknown. Here, we demonstrate lysosomal polyamine accumulation in ATP13A2 knockout (KO) cell lines. Primary polyamine storage caused secondary storage of lysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP) and age-dependent increase in the β-glucocerebrosidase (GCase) substrate, glucosylsphingosine, in Atp13a2 KO brains. Polyamine accumulation inhibited lysosomal GCase activity in cells and this was reversed by lysosome reacidification or BMP supplementation. A liposome-based GCase assay utilizing physiological substrates demonstrated dose-dependent inhibition of BMP-stimulated GCase activity by polyamines, in part via a pH-independent, electrostatics-based mechanism. Therefore, excess polyamine compromises lysosomes by disrupting pH and electrostatic interactions between GCase and BMP enabling efficient substrate hydrolysis, potentially clarifying their pathogenic mechanisms, and suggesting convergence on PD-relevant pathways.
Institute:	Denali Therapeutics
Last Name:	Suh
First Name:	Jung
Address:	161 Oyster Point Blvd, South San Francisco, California, 94080, USA
Email:	suh@dnli.com
Phone:	+1 6507973837

Subject:

Subject ID:	SU004204
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	Genotype	Treatment	Sample source	Sample Type
SA469288	HSA-000043797	ATP13A2 KO	22 6 PG	HAP1	whole cell lysate
SA469289	HSA-000043798	ATP13A2 KO	22 6 PG	HAP1	whole cell lysate
SA469290	HSA-000043799	ATP13A2 KO	22 6 PG	HAP1	whole cell lysate
SA469291	HSA-000043792	ATP13A2 KO	DOPC	HAP1	whole cell lysate
SA469292	HSA-000043793	ATP13A2 KO	DOPC	HAP1	whole cell lysate
SA469293	HSA-000043791	ATP13A2 KO	DOPC	HAP1	whole cell lysate
SA469318	HSA-000038935	ATP13A2 KO	none	HAP1	lysosome
SA469319	HSA-000038937	ATP13A2 KO	none	HAP1	lysosome
SA469320	HSA-000038936	ATP13A2 KO	none	HAP1	lysosome
SA469321	HSA-000043632	ATP13A2 KO	none	HAP1	whole cell lysate
SA469322	HSA-000043634	ATP13A2 KO	none	HAP1	whole cell lysate
SA469323	HSA-000043633	ATP13A2 KO	none	HAP1	whole cell lysate
SA469324	HSA-000038931	ATP13A2 KO	none	HAP1	whole cell lysate
SA469325	HSA-000038930	ATP13A2 KO	none	HAP1	whole cell lysate
SA469326	HSA-000038929	ATP13A2 KO	none	HAP1	whole cell lysate
SA469327	HSA-000043635	ATP13A2 KO	none	HAP1	whole cell lysate
SA469294	HSA-000043738	ATP13A2 KO	PBS	HAP1	whole cell lysate
SA469295	HSA-000043777	ATP13A2 KO	PBS	HAP1	whole cell lysate
SA469296	HSA-000043739	ATP13A2 KO	PBS	HAP1	whole cell lysate
SA469297	HSA-000043737	ATP13A2 KO	PBS	HAP1	whole cell lysate
SA469298	HSA-000043776	ATP13A2 KO	PBS	HAP1	whole cell lysate
SA469299	HSA-000043778	ATP13A2 KO	PBS	HAP1	whole cell lysate
SA469300	HSA-000043743	ATP13A2 KO	Rim	HAP1	whole cell lysate
SA469301	HSA-000043745	ATP13A2 KO	Rim	HAP1	whole cell lysate
SA469302	HSA-000043744	ATP13A2 KO	Rim	HAP1	whole cell lysate
SA469303	HSA-000043790	ATP13A2 KO	SPM + 22 6 PG	HAP1	whole cell lysate
SA469304	HSA-000043788	ATP13A2 KO	SPM + 22 6 PG	HAP1	whole cell lysate
SA469305	HSA-000043789	ATP13A2 KO	SPM + 22 6 PG	HAP1	whole cell lysate
SA469306	HSA-000043784	ATP13A2 KO	SPM + DOPC	HAP1	whole cell lysate
SA469307	HSA-000043783	ATP13A2 KO	SPM + DOPC	HAP1	whole cell lysate
SA469308	HSA-000043782	ATP13A2 KO	SPM + DOPC	HAP1	whole cell lysate
SA469309	HSA-000043746	ATP13A2 KO	SPM + Rim	HAP1	whole cell lysate
SA469310	HSA-000043748	ATP13A2 KO	SPM + Rim	HAP1	whole cell lysate
SA469311	HSA-000043747	ATP13A2 KO	SPM + Rim	HAP1	whole cell lysate
SA469312	HSA-000043781	ATP13A2 KO	SPM	HAP1	whole cell lysate
SA469313	HSA-000043742	ATP13A2 KO	SPM	HAP1	whole cell lysate
SA469314	HSA-000043740	ATP13A2 KO	SPM	HAP1	whole cell lysate
SA469315	HSA-000043741	ATP13A2 KO	SPM	HAP1	whole cell lysate
SA469316	HSA-000043780	ATP13A2 KO	SPM	HAP1	whole cell lysate
SA469317	HSA-000043779	ATP13A2 KO	SPM	HAP1	whole cell lysate
SA469328	HSA-000040326	NA	NA	HAP1	lysosome
SA469329	HSA-000040327	NA	NA	HAP1	lysosome
SA469330	HSA-000038939	NA	NA	HAP1	lysosome
SA469331	HSA-000040325	NA	NA	HAP1	lysosome
SA469332	HSA-000038938	NA	NA	HAP1	whole cell lysate
SA469333	HSA-000040322	NA	NA	HAP1	whole cell lysate
SA469334	HSA-000040323	NA	NA	HAP1	whole cell lysate
SA469335	HSA-000043818	NA	NA	HAP1	whole cell lysate
SA469336	HSA-000040324	NA	NA	HAP1	whole cell lysate
SA469337	HSA-000043819	NA	NA	HAP1	whole cell lysate
SA469338	HSA-000043722	NA	NA	HAP1	whole cell lysate
SA469339	HSA-000043816	NA	NA	HAP1	whole cell lysate
SA469340	HSA-000043812	NA	NA	HAP1	whole cell lysate
SA469341	HSA-000043813	NA	NA	HAP1	whole cell lysate
SA469342	HSA-000043814	NA	NA	HAP1	whole cell lysate
SA469343	HSA-000043815	NA	NA	HAP1	whole cell lysate
SA469344	HSA-000043817	NA	NA	HAP1	whole cell lysate
SA469345	HSA-000043775	WT	22 6 PG	HAP1	whole cell lysate
SA469346	HSA-000043774	WT	22 6 PG	HAP1	whole cell lysate
SA469347	HSA-000043773	WT	22 6 PG	HAP1	whole cell lysate
SA469348	HSA-000043768	WT	DOPC	HAP1	whole cell lysate
SA469349	HSA-000043769	WT	DOPC	HAP1	whole cell lysate
SA469350	HSA-000043767	WT	DOPC	HAP1	whole cell lysate
SA469375	HSA-000038934	WT	none	HAP1	lysosome
SA469376	HSA-000038932	WT	none	HAP1	lysosome
SA469377	HSA-000038933	WT	none	HAP1	lysosome
SA469378	HSA-000043626	WT	none	HAP1	whole cell lysate
SA469379	HSA-000038927	WT	none	HAP1	whole cell lysate
SA469380	HSA-000038926	WT	none	HAP1	whole cell lysate
SA469381	HSA-000043624	WT	none	HAP1	whole cell lysate
SA469382	HSA-000038928	WT	none	HAP1	whole cell lysate
SA469383	HSA-000043625	WT	none	HAP1	whole cell lysate
SA469384	HSA-000043627	WT	none	HAP1	whole cell lysate
SA469351	HSA-000043726	WT	PBS	HAP1	whole cell lysate
SA469352	HSA-000043725	WT	PBS	HAP1	whole cell lysate
SA469353	HSA-000043727	WT	PBS	HAP1	whole cell lysate
SA469354	HSA-000043753	WT	PBS	HAP1	whole cell lysate
SA469355	HSA-000043754	WT	PBS	HAP1	whole cell lysate
SA469356	HSA-000043752	WT	PBS	HAP1	whole cell lysate
SA469357	HSA-000043731	WT	Rim	HAP1	whole cell lysate
SA469358	HSA-000043732	WT	Rim	HAP1	whole cell lysate
SA469359	HSA-000043733	WT	Rim	HAP1	whole cell lysate
SA469360	HSA-000043766	WT	SPM + 22 6 PG	HAP1	whole cell lysate
SA469361	HSA-000043765	WT	SPM + 22 6 PG	HAP1	whole cell lysate
SA469362	HSA-000043764	WT	SPM + 22 6 PG	HAP1	whole cell lysate
SA469363	HSA-000043759	WT	SPM + DOPC	HAP1	whole cell lysate
SA469364	HSA-000043758	WT	SPM + DOPC	HAP1	whole cell lysate
SA469365	HSA-000043760	WT	SPM + DOPC	HAP1	whole cell lysate
SA469366	HSA-000043736	WT	SPM + Rim	HAP1	whole cell lysate
SA469367	HSA-000043734	WT	SPM + Rim	HAP1	whole cell lysate
SA469368	HSA-000043735	WT	SPM + Rim	HAP1	whole cell lysate
SA469369	HSA-000043728	WT	SPM	HAP1	whole cell lysate
SA469370	HSA-000043730	WT	SPM	HAP1	whole cell lysate
SA469371	HSA-000043756	WT	SPM	HAP1	whole cell lysate
SA469372	HSA-000043729	WT	SPM	HAP1	whole cell lysate
SA469373	HSA-000043757	WT	SPM	HAP1	whole cell lysate
SA469374	HSA-000043755	WT	SPM	HAP1	whole cell lysate

Showing results 1 to 97 of 97

Showing results 1 to 97 of 97

Collection:

Collection ID:	CO004197
Collection Summary:	Human HAP1 parental WT and ATP13A2 KO cells (~25,000) were washed 1x with 0.9% sodium chloride and harvested for metabolite and lipid extraction. Lysosomal fractions were isolated from HAP1 parental WT and ATP13A2 KO cells following endocytosis-mediated labeling using superparamagnetic iron oxide nanoparticles (SPION). HAP1 cells were seeded in 3X 10cm tissue culture dishes and allowed to grow till ~75% confluency at which time they were treated for 24 h with 10% (v/v) dextran-magnetite solution (DexoMAG40, Liquids Research Ltd.). The cells were rinsed with warmed 1X PBS, pH 7.4 (Gibco; # 10010023) to remove residual extracellular dextran-magnetite and re-supplemented with fresh culture medium for at least 4 h to allow for specific labeling of lysosomes and late endosomes. Cells were harvested by scraping with ice cold 1X PBS and centrifugation at 300 x g for 5 min at 4°C; resuspended in isolation buffer (250mM sucrose, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 1 mM DTT, cOmplete Protease Inhibitor (Roche; #04693132001)), and replicates pooled by centrifugation into 1 mL lysis buffer. Cells were lysed using a 27G needle for 20 strokes, followed by centrifugation of the cell lysate at 600 x g for 10 min at 4°C. Post-nuclear supernatant (PNS) was collected, and pellet was resuspended in 1 mL isolation buffer followed by another round of needle lysis and PNS collection to capture maximum lysosomes. Pooled supernatants from both rounds of needle lysis were then loaded onto LS columns (Miltenyi Biotec; #130-042-401), attached to Quadro MACS magnet, that were already pre equilibrated with chilled isolation buffer. Flowthrough was collected after passing the PNS through the column by gravity flow and passed through the column again to increase retention of unbound labeled lysosomes. Finally, the columns were washed three times with isolation buffer to eliminate other membrane-bound organelle contaminants, and magnetically bound lysosomes eluted twice into 0.5 mL isolation buffer. The elutes were diluted with 1 mL 1X PBS, centrifuged at 21,000 x g for 40 min at 4°C. The lysosomal pellet was resuspended in 200 µL isolation buffer and harvested for metabolite and lipid extraction.
Sample Type:	HAP1 cells
Storage Conditions:	-80℃
Collection Vials:	Lobind 1.5 mL Eppendorf tubes
Storage Vials:	Lobind 1.5 mL Eppendorf tubes

Treatment:

Treatment ID:	TR004213
Treatment Summary:	Human HAP1 parental WT and ATP13A2 KO cells (Horizon Discovery; WT: C631; ATP13A2 KO: HZGHC003547c002) were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco; #12440053) supplemented with 10% fetal bovine serum (VWR Seradigm; #97068-085) and 1% Penicillin/Streptomycin (Gibco; #15140122). No treatment.Cells were treated for 20 hours with PBS, Rimeporide(Rim), Spermine (SPM), or a combination of Spermine (SPM) + Rimeporide(Rim), followed by metabolite extraction. Cells were treated for 20 hours with PBS , Spermine (SPM), 18:1 (Δ9-Cis) PC (DOPC), 22:6 PG, a combination of Spermine(SPM) + DOPC, or a combination of Spermine (SPM) + 22:6 PG,followed by metabolite extraction. NOTE: To evaluate whether BMP can rescue spermine-dependent inhibition, WT HAP1 cells were treated with spermine and supplemented with PG(22:6/22:6), a structural isomer and precursor of BMP(22:6/22:6). PG (22:6/22:6) was supplemented due to its lower cost and greater availability, relying on its established rapid conversion to BMP in lysosomes (see 10.1016/j.bbalip.2021.158916).

Treatment ID:

TR004213

Treatment Summary:

Human HAP1 parental WT and ATP13A2 KO cells (Horizon Discovery; WT: C631; ATP13A2 KO: HZGHC003547c002) were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco; #12440053) supplemented with 10% fetal bovine serum (VWR Seradigm; #97068-085) and 1% Penicillin/Streptomycin (Gibco; #15140122). No treatment.Cells were treated for 20 hours with PBS, Rimeporide(Rim), Spermine (SPM), or a combination of Spermine (SPM) + Rimeporide(Rim), followed by metabolite extraction. Cells were treated for 20 hours with PBS , Spermine (SPM), 18:1 (Δ9-Cis) PC (DOPC), 22:6 PG, a combination of Spermine(SPM) + DOPC, or a combination of Spermine (SPM) + 22:6 PG,followed by metabolite extraction. NOTE: To evaluate whether BMP can rescue spermine-dependent inhibition, WT HAP1 cells were treated with spermine and supplemented with PG(22:6/22:6), a structural isomer and precursor of BMP(22:6/22:6). PG (22:6/22:6) was supplemented due to its lower cost and greater availability, relying on its established rapid conversion to BMP in lysosomes (see 10.1016/j.bbalip.2021.158916).

Sample Preparation:

Sampleprep ID:	SP004210
Sampleprep Summary:	Cells (~25,000) and lysosomal fractions (100 µg) were directly lysed and extracted in 150 µL methanol containing stable-isotope internal standards for 20 min at 4°C with shaking. The supernatant fraction was transferred to a 96-well sample collection plate (Waters; #186005837) following a 14,000 x g spin at 4°C for 20 min from which the supernatant was distributed for the LC-MS/MS analyses.
Processing Storage Conditions:	On ice
Extract Storage:	-20℃

Chromatography:

Chromatography ID:	CH005095
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Imtakt Intrada Organic Acid (150 × 2 mm, 3 um)
Column Temperature:	60
Flow Gradient:	0.0-1.0 min at 0% B; 1.0-7.0 min to 100% B; 7.1 at 0% B; and 7.1-10 min at 0% B.
Flow Rate:	0.20 mL/min
Sample Injection:	5
Solvent A:	10% acetonitrile/90% water; 0.1% formic acid
Solvent B:	10% acetonitrile/90% water; 100mM ammonium formate
Chromatography Type:	Ion exchange

Chromatography ID:	CH005096
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	55
Flow Gradient:	0.0-8.0 min from 45% B to 99% B, 8.0-9.0 min at 99% B, 9.0-9.1 min to 45% B, and 9.1-10.0 min at 45% B.
Flow Rate:	0.25 mL/min
Sample Injection:	5
Solvent A:	60% acetonitrile/40% water; with 10 mM ammonium formate; 0.1% formic acid
Solvent B:	90% isopropyl alcohol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH005097
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Advanced Materials Technology HALO HILIC column (150 x 3.0 mm, 2um); #91813
Column Temperature:	45
Flow Gradient:	0.0–2.0 min, 100% B; 2.1 min, 95% B; 4.5 min, 85% B; held at 85% B until 6.0 min; 6.1 min, 0% B; held at 0% B until 8.5 min
Flow Rate:	0.45 mL/min
Sample Injection:	8
Solvent A:	92.5% acetonitrile/5% isopropanol/2.5% water; 5 mM ammonium formate; 0.5% formic acid
Solvent B:	92.5% water/5% isopropanol/2.5% acetonitrile; 5 mM ammonium formate; 0.5% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH005098
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	55
Flow Gradient:	0.0-8.0 min from 45% B to 99% B, 8.0-9.0 min at 99% B, 9.0-9.1 min to 45% B, and 9.1-10.0 min at 45% B.
Flow Rate:	0.25 mL/min
Sample Injection:	5
Solvent A:	60% acetonitrile/40% water; 10 mM ammonium acetate; 0.1% acetic acid
Solvent B:	90% isopropyl alcohol/10% acetonitrile; 10 mM ammonium acetate; 0.1% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH005099
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	55
Flow Gradient:	0.00–0.20 min at 20% B, 0.20–2.50 min at 20% B, 2.50–3.50 min from 20% B to 95% B, 3.50–4.40 min at 95% B, 4.40–4.50 min to 20% B, and 4.50–6.00 min at 20% B
Flow Rate:	0.40 mL/min
Sample Injection:	3
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN006707
Analysis Type:	MS
Chromatography ID:	CH005095
Num Factors:	22
Num Metabolites:	60
Units:	normalized peak area

Analysis ID:	AN006708
Analysis Type:	MS
Chromatography ID:	CH005096
Num Factors:	22
Num Metabolites:	216
Units:	normalized peak area

Analysis ID:	AN006709
Analysis Type:	MS
Chromatography ID:	CH005097
Num Factors:	22
Num Metabolites:	39
Units:	normalized peak area

Analysis ID:	AN006710
Analysis Type:	MS
Chromatography ID:	CH005098
Num Factors:	22
Num Metabolites:	119
Units:	normalized peak area

Analysis ID:	AN006711
Analysis Type:	MS
Chromatography ID:	CH005099
Num Factors:	22
Num Metabolites:	15
Units:	normalized peak area