PXD048696-2

PXD048696 is an original dataset announced via ProteomeXchange.

Title	Two step enrichment facilitates background reduction for proteomic analysis of lysosomes
Description	Lysosomes comprise the main degradative compartment of almost all mammalian cells and are involved in various cellular functions, most of which are catalyzed by the lysosomal proteome. Lysosomal proteins are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance, and to enable profiling of lysosomal content, lysosomes are frequently enriched, for which currently two approaches are most commonly used: superparamagnetic nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). We investigated the effects of both approaches on the lysosomal proteome through enrichment of lysosomes with the respective other approach proteomic analyses. For SPIONs treatment, we identified mainly an effect on cellular iron homeostasis, while changes of the lysosomal proteome were moderate. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially related to alterations of membrane proteins and such involved in protein trafficking. Furthermore, we established a combined strategy consisting of sequential lysosome enrichment with SPIONs and TMEM-IPs. This approach enabled increased purity of lysosome enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow through and eluate fractions, further characterization of the individual approaches’ properties.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:52:02.900.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Sara Bonini
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidoethyl-L-cysteine; acetylated residue; monohydroxylated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2024-01-18 13:21:09	ID requested
1	2024-08-01 02:44:55	announced
⏵ 2	2024-10-22 06:52:03	announced	2024-10-22: Updated project metadata.

10.1021/acs.jproteome.4c00053;

Bonini S, Winter D, Two-Step Enrichment Facilitates Background Reduction for Proteomic Analysis of Lysosomes. J Proteome Res, 23(8):3393-3403(2024) [pubmed]

submitter keyword: Lysosomes

Lysosome enrichment

Superparamagnetic iron oxide particles (SPIONs)

TMEM192, mass spectrometry

proteomics

organelles, HEK 293

Dominic Winter
contact affiliation	Institute for Biochemistry and Molecular Biology, Medical Faculty, University of Bonn, Bonn 53115, Germany
contact email	dominic.winter@uni-bonn.de
lab head
Sara Bonini
contact affiliation	Institute of Biochemistry and Molecular Biology
contact email	sarab@uni-bonn.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD048696
     1. Label: PRIDE project
     2. Name: Two step enrichment facilitates background reduction for proteomic analysis of lysosomes