Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale

Martin Steger,Markus Ralser,Mattias Backman,Stefan Müller,Phillip Ihmor,Vadim Demichev,Henrik Daub,Uli Ohmayer

doi:10.1038/s41467-021-25454-1

Abstract

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput.

Highlights

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling
Despite its good performance in regular proteomic analyses[14,15], sodium deoxycholate (SDC)based protein extraction has only recently been explored for ubiquitinomics applications[16]
After tryptic digestion of proteins and immunoaffinity purification of K-GG remnant peptides, we acquired the data in data-dependent acquisition (DDA) mode

Summary

Introduction

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. We present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with dataindependent acquisition (DIA)-MS and neural network-based data processing optimized for ubiquitinomics. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput. While early studies on ubiquitinated proteins were conducted on a target-by-target basis, mass spectrometry (MS)-based proteomics has facilitated global ubiquitin signaling profiling, such as B-cell receptor signaling[6,7]. By introducing a sodium deoxycholate (SDC)-based lysis protocol, and exploiting dataindependent acquisition mass spectrometry (DIA-MS) coupled to deep neural network-based data processing, we boost reproducibility, identification numbers, and quantitative accuracy. Combining the profiles of ubiquitinated peptides with their corresponding protein abundances allows us to define putative USP7 targets with high confidence, and to distinguish regulatory ubiquitination leading to protein degradation from non-degradative events

Methods

Results

Conclusion