License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: October 08, 2024
Last Modified: October 31, 2024
Collection Integer ID: 111342
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000282
ASAP
Grant ID: ASAP-025160
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The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Lysosomal storage diseases (LSDs) comprise ~50 monogenic diseases display accumulation of cellular material in lysosomes and associated defects in lysosomal function, but systematic lipid-proteome phenotyping remains challenging. Here, we report a sensitive nanoflow-based multi-omic single-shot technology (nMOST) workflow allowing simultaneous quantification of proteomes and lipidomes. Benchmarking nMOST using LSD mutants linked with cholesterol trafficking revealed aberrant accumulation of autophagy receptors and ferritinophagy cargo, which correlated with accumulation of lyso-phosphatidylcholine species and multi-lamellar membrane structures visualized by cryo-electron-tomography, especially in NPC2-/- cells. Ferritinophagy defects correlated with loss of mitochondrial cristae and iron-sulfur cluster-containing electron transport chain complexes that could be rescued by extracellular iron. To further demonstrate the value of nMOST, we profiled more than two dozen diverse LSD mutant cell lines. We provide an accompanying web-portal resource of lipid-protein correlations across the entire dataset and demonstrate how the resource can be used to reveal common and distinct molecular phenotypes.
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Neurogenesis with iron supplementation and label-free nDIA proteomics
Version 1
Felix Kraus
Protocol references
Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST
Felix Kraus, Yuchen He, Sharan Swarup, Katherine A. Overmyer, Yizhi Jiang, JohannBrenner, Cristina Capitanio, Anna Bieber, Annie Jen, Nicole M. Nightingale, Benton J.Anderson, Chan Lee, Joao A. Paulo, Ian R. Smith, Jürgen M. Plitzko, Steven P. Gygi, Brenda A. Schulman, Florian Wilfling, Joshua J. Coon, J. Wade Harper
