Proteomics

Dataset Information

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Comprehensive proteomic analysis of mouse embryonic fibroblast lysosomes by mass spectrometry


ABSTRACT: The lysosome is the main degradative organelle in eukaryotic cells. It is involved in diverse cellular functions from intracellular digestion to metabolic signaling. Despite various efforts to enrich for lysosomes and analyze their proteome with mass spectrometry based proteomics approaches, no systematic evaluation of sample preparation parameters for the analysis of lysosome enriched fractions has been performed. Using samples enriched for lysosomes with paramagnetic nanoparticles, we conducted a systematic evaluation of the protocol for the analysis of the lysosomal proteome. We compared centrifugation and protein precipitation for the concentration of lysosome enriched fractions and tested four different digestion methods in combination with each of these approaches. This included filter aided sample preparation (FASP), in-gel digestion, and in solution digestion using either RapiGest or Urea. Furthermore, we evaluated three formats for sample desalting, four gradient lengths for LC-MSMS analysis of unfractionated samples, as well as fractionation by strong anion exchange tip columns into three or six fractions. Using the combined data, we generated a draft map of lysosomal proteome for mouse embryonic fibroblasts as well as a spectral library for the analysis of lysosomes by data independent acquisition (DIA) and evaluated different LC gradient lengths for DIA.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Jasjot Singh  

LAB HEAD: Dominic Winter

PROVIDER: PXD014576 | Pride | 2020-02-17

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
3Fraction.msf Msf
3Fraction_R1_F1.raw Raw
3Fraction_R1_F2.raw Raw
3Fraction_R1_F3.raw Raw
3Fraction_R2_F1.raw Raw
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