Work

An integrated workflow for quantitative analysis of the newly synthesized proteome

Year: 2023

Type: article

Abstract: The analysis of proteins that are newly synthesized upon a cellular perturbation can provide detailed insight into the proteomic response that is elicited by specific cues. This can be investigated by pulse-labeling of cells with clickable and stable-isotope-coded amino acids for the enrichment and mass spectrometric characterization of newly synthesized proteins (NSPs), however convoluted protocols prohibit their routine application. Here we report the optimization of multiple steps in sample preparation, mass spectrometry and data analysis, and we integrate them into a semi-automated workflow for the quantitative analysis of the newly synthesized proteome (QuaNPA). Reduced input requirements and data-independent acquisition (DIA) enable the analysis of triple-SILAC-labeled NSP samples, with enhanced throughput while featuring high quantitative accuracy. We apply QuaNPA to investigate the time-resolved cellular response to interferon-gamma (IFNg), observing rapid induction of targets 2 h after IFNg treatment. QuaNPA provides a powerful approach for large-scale investigation of NSPs to gain insight into complex cellular processes.

Source: Nature Communications

DOI: 10.1038/s41467-023-43919-3

Authors: Toman Borteçen, Torsten Müller, Jeroen Krijgsveld

Institutions: German Cancer Research Center, Heidelberg University

Language: English

Topic: Advanced Proteomics Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: gold

Estimated APC paid: $6,290

Funder: Bundesministerium für Bildung und Forschung