Cargo and lipid profiling of isolated autophagosomes upon basal autophagy, starvation, and proteasome inhibition Version 1
External LinkAutophagy is responsible for degradation of an extensive portfolio of cytosolic cargoes that are engulfed in autophagosomes to facilitate their transport to lysosomes. Besides basal autophagy, which constantly degrades cellular material, the pathway is dynamically altered by different conditions, resulting in enhanced autophagosome formation and cargo turnover. The extensive profile of autophagosome content as well as the phospholipid composition of human autophagosome membranes remains elusive. Here, we introduce a novel FACS-based approach for isolation of native autophagosomes and confirm the quality and specificity of the preparations. Employing quantitative proteomics and lipidomics, we obtained a profound cargo and lipid profile of autophagosomes isolated upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. This resource will allow to identify the impact of different autophagy conditions on the degradation of distinct proteins and to dissect the phospholipid composition of the human autophagosome membrane in detail.
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Created: 9th Jul 2024 at 13:38
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Version 1 (earliest) Created 9th Jul 2024 at 13:38 by Rainer Malik
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Institutions: LMU Klinikum
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Submitter: Rainer Malik
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Assays: Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Affinity purification coupled with mass spectrometry proteomics (human), Bottom-up proteomics (human), Bottom-up proteomics (mouse), Bottom-up proteomics (mouse), Bottom-up proteomics (mouse), Bottom-up proteomics (mouse), Gel-based experiment (human), Phosphoproteomics / Bottom-up proteomics (mouse), Proximity-proteomics-based autophagosome content profiling (human), SWATH MS (human), SWATH MS (human, mouse), SWATH MS (mouse), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human), Shotgun proteomics (human, mouse), Shotgun proteomics (human, mouse), Shotgun proteomics (human, mouse), Shotgun proteomics (human, mouse), Shotgun proteomics (macaque), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (mouse), Shotgun proteomics (rat), Untargeted Proteomics (mouse)
Autophagy is responsible for degradation of an extensive portfolio of cytosolic cargoes that are engulfed in autophagosomes to facilitate their transport to lysosomes. Besides basal autophagy, which constantly degrades cellular material, the pathway is dynamically altered by different conditions, resulting in enhanced autophagosome formation and cargo turnover. The extensive profile of autophagosome content as well as the phospholipid composition of human autophagosome membranes remains elusive. ...
Sample preparation for LC-MS/MS Eight million of Cy3/5-positive autophagosomes (roughly 10ug of proteins) were denatured with 2% sodium deoxycholate, 50 mM Tris-HCl pH 8.5, 2.5 mM TCEP, 10 mM chloroacetamide at 95°C for 10 min. Lysates were prepared with in-StageTip (iST) processing method for LC-MS/MS as previously described by Kulak et al. 2014. Briefly, Proteins were digested overnight at 37°C with 1 volume of 50mM Tris-HCl pH 8.5 containing LysC (Wako Chemicals) at 1:100 (w/w) ratio and Trypsin ...
Submitter: Rainer Malik
Assay type: Proteomics
Technology type: Technology Type
Investigation: Proteomics (Published)
