Quantitative analysis of global protein stability rates in tissues

Daniel B Mcclatchy,John R Yates,Mathieu Lavallée-Adam,Salvador Martínez-Bartolomé,Yu Gao

doi:10.1038/s41598-020-72410-y

Daniel B Mcclatchy, John R Yates + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-72410-y

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Abstract

Protein degradation is an essential mechanism for maintaining proteostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases. We present a new technique, QUAD (Quantification of Azidohomoalanine Degradation), to analyze the global degradation rates in tissues using a non-canonical amino acid and mass spectrometry. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability. Within a tissue, different organelles and protein functions were enriched with different stability patterns. QUAD analysis demonstrated that protein stability is enhanced with age in the brain but not in the liver. Overall, QUAD allows the first global quantitation of protein stability rates in tissues, which will allow new insights and hypotheses in basic and translational research.

Highlights

Protein degradation is an essential mechanism for maintaining proteostasis in response to internal and external perturbations
We propose to use azidohomoalanine (AHA) to directly quantitate protein stability rates in tissues using pulse-chase labeling coupled with MS, which will provide better temporal resolution than the extreme long-lived proteins (ELLP) strategy
AHA is a non-canonical amino acid(ncAA) that can be inserted into proteins in vivo because it is accepted by the endogenous methionine tRNA synthetase

Summary

Introduction

Protein degradation is an essential mechanism for maintaining proteostasis in response to internal and external perturbations. Global protein stability rates, are generally measured a “pulse-chase” experiment, where proteins are labeled or tagged and quantitated by the loss of protein signal with time using immunoblots, fluorescence, or mass spectrometry(MS)[4,5,6,7,8,9,10,11] These techniques are primarily used on cultured cells because they are difficult to employ in tissue. Over the decade of biological studies employing AHA, no toxicity has been reported, suggesting little to no perturbation to native protein characteristics This mostly likely stems from the fact that AHA structure is so remarkably similar to methionine that it interacts with the endogenous tRNA synthetase. We Quantification of AHA Degradation (QUAD) in labeled proteins to measure protein stability rates in tissues

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