An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping.

Journal

bioRxiv : the preprint server for biology
Titre abrégé: bioRxiv
Pays: United States
ID NLM: 101680187

Informations de publication

Date de publication:
12 Apr 2023
Historique:
pubmed: 24 4 2023
medline: 24 4 2023
entrez: 24 04 2023
Statut: epublish

Résumé

Proximity labeling (PL) coupled with mass spectrometry has emerged as a powerful technique to map proximal protein interactions in living cells. Large-scale sample processing for proximity proteomics necessitates a high-throughput workflow to reduce hands-on time and increase quantitative reproducibility. To address this issue, we developed a scalable and automated PL pipeline, including generation and characterization of monoclonal cell lines, automated enrichment of biotinylated proteins in a 96-well format, and optimization of the quantitative mass spectrometry (MS) acquisition method. Combined with data-independent acquisition (DIA) MS, our pipeline outperforms manual enrichment and data-dependent acquisition (DDA) MS regarding reproducibility of protein identification and quantification. We apply the pipeline to map subcellular proteomes for endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, using serotonin receptor (5HT

Identifiants

DOI: 10.1101/2023.04.11.536358 PMID: 37090610 PMC: PMC10120663
pubmed: 37090610
doi: 10.1101/2023.04.11.536358
pmc: PMC10120663
pii:
doi:

Types de publication

Preprint

Langues

eng

Subventions

Organisme : NHLBI NIH HHS
ID : P01 HL146366
Pays : United States

Déclaration de conflit d'intérêts

Competing Interest Statement The Krogan Laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche. Nevan Krogan has consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics and Interline Therapeutics. He is a shareholder in Tenaya Therapeutics, Maze Therapeutics and Interline Therapeutics, and a financially compensated Scientific Advisory Board Member for GEn1E Lifesciences, Inc.

Auteurs

Xiaofang Zhong (X)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Qiongyu Li (Q)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Benjamin J Polacco (BJ)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Trupti Patil (T)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Jeffrey F DiBerto (JF)

Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Rasika Vartak (R)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Jiewei Xu (J)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Aaron Marley (A)

Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, CA 94158, USA.

Helene Foussard (H)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Bryan L Roth (BL)

Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Manon Eckhardt (M)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Mark Von Zastrow (M)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.
Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, CA 94158, USA.

Nevan J Krogan (NJ)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Ruth Hüttenhain (R)

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Classifications MeSH