Multi cell line analysis of lysosomal proteomes reveals unique features and novel lysosomal proteins

Akter, Fatema; Ponnaiyan, Srigayatri; Koegler-Mohrbacher, Bianca; Bleibaum, Florian; Damme, Markus; Renard, Bernhard Y.; Winter, Dominic

doi:10.1101/2020.12.21.423747

Cited by 10 publications

(15 citation statements)

References 65 publications

(83 reference statements)

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“…For the unbiased characterization of large numbers of proteins, mass spectrometry (MS)-based proteomics is currently the method of choice, as it allows for the identification, quantification, and characterization of thousands of proteins from a given sample [12]. To date,~740 proteins have been assigned in one way or the other to lysosomes,~300 of which are either located in the lysosomal lumen, at the lysosomal surface, or directly interact with it [13].…”

Section: Introductionmentioning

confidence: 99%

Targeted Quantification of the Lysosomal Proteome in Complex Samples

et al. 2021

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In eukaryotic cells, lysosomes play a crucial role in the breakdown of a variety of components ranging from small molecules to complex structures, ascertaining the continuous turnover of cellular building blocks. Furthermore, they act as a regulatory hub for metabolism, being crucially involved in the regulation of major signaling pathways. Currently, ~450 lysosomal proteins can be reproducibly identified in a single cell line by mass spectrometry, most of which are low-abundant, restricting their unbiased proteomic analysis to lysosome-enriched fractions. In the current study, we applied two strategies for the targeted investigation of the lysosomal proteome in complex samples: data-independent acquisition (DIA) and parallel reaction monitoring (PRM). Using a lysosome-enriched fraction, mouse embryonic fibroblast whole cell lysate, and mouse liver whole tissue lysate, we investigated the capabilities of DIA and PRM to investigate the lysosomal proteome. While both approaches identified and quantified lysosomal proteins in all sample types, and their data largely correlated, DIA identified on average more proteins, especially for lower complex samples and longer chromatographic gradients. For the highly complex tissue sample and shorter gradients, however, PRM delivered a better performance regarding both identification and quantification of lysosomal proteins. All data are available via ProteomeXchange with identifier PXDD023278.

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Section: Introductionmentioning

confidence: 99%

Targeted Quantification of the Lysosomal Proteome in Complex Samples

et al. 2021

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“…Using a non-cross-linked fraction of each sample, we acquired an LC-MS/MS reference dataset. In total, we identified 4,181 proteins, of which 474 were assigned the term "lysosome" based on GO terms and UniProt classifiers in >3 runs, indicating an excellent performance of lysosome enrichment (Akter, 2020). To assess the quantitative distribution of lysosomal proteins in our samples, we utilized these data to estimate absolute protein abundances by intensity-based absolute quantification (iBAQ) (Schwanhausser et al, 2011).…”

Section: Cross-linking Mass Spectrometry Analysis Of Lysosome Enriche...mentioning

confidence: 99%

“…As lysosomal proteins are expressed at a dynamic abundance range encompassing three orders of magnitude (Akter, 2020), we further correlated CSMs, peptide spectral matches (PSMs), and iBAQ values. Even though higher abundant proteins tended to yield more CSMs, we did not identify a strong correlation between cross-link identification and protein abundance, showing that our dataset also covered proteins of low expression levels (Figure 1H, Figure S1I).…”

Section: Cross-linking Mass Spectrometry Analysis Of Lysosome Enriche...mentioning

confidence: 99%

“…L) Categorization of the 1,415 CSMs identified for 68 lysosomal proteins. Proteins were assigned to previously defined categories of lysosomal proteins (Akter, 2020) A-C) Protein-protein interaction networks based on cross-links identified between two different proteins (inter-links, 1,023 in total). Lysosomal proteins are highlighted as large blue filled circles while non-lysosomal proteins are depicted as small grey dots.…”

Section: D)mentioning

confidence: 99%

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Cross-linking of the Endolysosomal System Reveals Flotillin Structures and Putative Cargo

Singh

Elhabashy

Muthukottiappan

et al. 2022

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Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes, but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyzed lysosomes and early endosomes. Based on the identification of 5,376 cross-links, we investigated protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and heterodimeric/-multimeric structures of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identified >300 proteins presenting putative cargo, and confirm the latrophilin family of adhesion G protein-coupled receptors as substrates for flotillin-dependent endocytosis.

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“…UC n and UC@CB [7] showed very low cytotoxicity on HeLa; see Figure 3. The higher cytotoxicity observed in HeLa cells compared to the EAhy 926 endothelial cells can be explained by the higher content of lysosomes of the HeLa cells [38]. Since UCNPs can be dissolved in an acidic environment, cells with a higher lysosome content are expected to lead to higher levels of F -, Na + , Y 3+ , and Ln 3+ ions with potential cytotoxic effects.…”

Section: Cytotoxicitymentioning

confidence: 99%

Initial Biological Assessment of Upconversion Nanohybrids

et al. 2021

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Nanoparticles for medical use should be non-cytotoxic and free of bacterial contamination. Upconversion nanoparticles (UCNPs) coated with cucurbit[7]uril (CB[7]) made by combining UCNPs free of oleic acid, here termed bare UCNPs (UCn), and CB[7], i.e., UC@CB[7] nanohybrids, could be used as photoactive inorganic-organic hybrid scaffolds for biological applications. UCNPs, in general, are not considered to be highly toxic materials, but the release of fluorides and lanthanides upon their dissolution may cause cytotoxicity. To identify potential adverse effects of the nanoparticles, dehydrogenase activity of endothelial cells, exposed to various concentrations of the UCNPs, was determined. Data were verified by measuring lactate dehydrogenase release as the indicator of loss of plasma membrane integrity, which indicates necrotic cell death. This assay, in combination with calcein AM/Ethidium homodimer-1 staining, identified induction of apoptosis as main mode of cell death for both particles. The data showed that the UCNPs are not cytotoxic to endothelial cells, and the samples did not contain endotoxin contamination. Higher cytotoxicity, however, was seen in HeLa and RAW 264.7 cells. This may be explained by differences in lysosome content and particle uptake rate. Internalization of UCn and UC@CB[7] nanohybrids by cells was demonstrated by NIR laser scanning microscopy.

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Multi cell line analysis of lysosomal proteomes reveals unique features and novel lysosomal proteins

Cited by 10 publications

References 65 publications

Targeted Quantification of the Lysosomal Proteome in Complex Samples

Targeted Quantification of the Lysosomal Proteome in Complex Samples

Cross-linking of the Endolysosomal System Reveals Flotillin Structures and Putative Cargo

Initial Biological Assessment of Upconversion Nanohybrids

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