Rapid and deep-scale ubiquitylation profiling for biology and translational research

Abstract

Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying 10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for largescale studies in primary tissue samples.

Ubiquitomics: An Overview and Future

“…Performing TMT labelling on the anti-K-GG coated beads after pulldown of the GG-modified peptides enables selective labelling of the peptide N-terminus and allows TMT contaminants to be washed away, increasing the number of identified ubiquitination sites relative to in-solution TMT labelling and reducing sample requirement to sub-milligram levels. This methodology is referred to as UbiFast [ 29 ].…”

“…There are now proteomic workflows that enable the analysis of multiple “PTMomes” in single experiments through sequential pulldowns of different modifications after trypsinisation. Measurement of acetylomes and phosphoproteomes from the same sample as the ubiquitome show the interplay between PTMs and how they act together to co-ordinate cellular events [ 29 , 30 , 31 , 32 ]. Ubiquitome datasets increasingly include a matching proteome [ 21 , 29 , 32 , 33 ].…”

“…Measurement of acetylomes and phosphoproteomes from the same sample as the ubiquitome show the interplay between PTMs and how they act together to co-ordinate cellular events [ 29 , 30 , 31 , 32 ]. Ubiquitome datasets increasingly include a matching proteome [ 21 , 29 , 32 , 33 ]. An increase in the MS quantitation value of a GG-modified peptide is more commonly explained by an increase in the abundance of its corresponding protein, so a matching proteome is necessary to tease apart differentially ubiquitinated (or otherwise modified) proteins and distinguish regulatory versus degradative ubiquitin modifications.…”

“…In the Protein Lysine Modification Database from 2017 there are 24,487 co-occurrences of acetylation and ubiquitination on the same residue [ 41 ]. Studies that profile both acetylation and ubiquitin sites have found a negative correlation between the two modifications on proteins [ 29 ]. This suggests that acetylation of a lysine residue could physically block ubiquitination at that site, this being particularly relevant in the case of epigenetic regulations [ 42 ].…”

“…The seminal UbiSite paper used up to 50 mg of protein starting material per condition in triplicate [ 21 ], and the CST PTMScan Ubiquitin Remnant Motif Kit recommends using 10–20 mg of protein input per pulldown. There have been titrations reported with varying amount of starting material [ 29 , 78 ] and new sample preparation methods that require less material [ 29 ]. For instance, the latest technology from CST, the PTMScan HS Ubiquitin/SUMO Remnant Motif kit (59322), requires only 1 mg of starting material by implementing SDS lysis and S-Trap columns in the workflow.…”