Mass Spectrometry-Based Proteome Analysis

Mass spectrometric analysis was performed as described (Itzhak et al., 2016 (link)), using a Thermo EASY-nLC 1000 HPLC coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific, Germany). HPLC gradient lengths varied for the different experiments. For analysis of whole proteomes, each of the SCX peptide fractions was analyzed with a 240 min gradient (24 h per sample in total). For the analysis of cytosol and fractions from the organellar maps, each sample was analyzed with a single 150 min gradient. Raw files were processed with MaxQuant software Version 1.6 (Cox and Mann, 2008 (link)), using the murine reference proteome (Swiss-Prot canonical and isoform data) database downloaded from UniProt (https://www.uniprot.org:443/).

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Kozik P., Gros M., Itzhak D.N., Joannas L., Heurtebise-Chrétien S., Krawczyk P.A., Rodríguez-Silvestre P., Alloatti A., Magalhaes J.G., Del Nery E., Borner G.H, & Amigorena S. (2020). Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity. Cell Reports, 32(2), 107905.

Publication 2020

Q Exactive HF Hybrid Quadrupole-Orbitrap

Cytosol Hplc Hybrid Isoform Maps Mass spectrometric Murine Organellar Peptide Proteome

Corresponding Organization :

Other organizations : Immunité et Cancer, MRC Laboratory of Molecular Biology, Max Planck Institute of Biochemistry

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Variable analysis

independent variables

HPLC gradient lengths

dependent variables

Mass spectrometric analysis results

control variables

Experimental procedure as described in Itzhak et al., 2016
Use of a Thermo EASY-nLC 1000 HPLC coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap
Processing of raw files with MaxQuant software Version 1.6
Use of the murine reference proteome (Swiss-Prot canonical and isoform data) database from UniProt

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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