UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research

Abstract

Protein ubiquitylation is involved in a plethora of cellular processes. Defects in the ubiquitin system are at the root of many acquired and hereditary diseases. 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 for quantifying ~10,000 ubiquitylation sites from as little as 500 ug peptide per sample from cells or tissue in a TMT10 plex in ca. 5 hr. High-field Asymmetric 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 large-scale studies in primary tissue samples.

Ubiquitylome Analysis Reveals a Central Role for the Ubiquitin-Proteasome System in Plant Innate Immunity

“…Furthermore, we should also emphasize the TUBE affinity approach likely binds polyubiquitylated proteins better than monoubiquitylated proteins and thus reduced the latter in our catalogs. One strategy to avoid this complication is to enrich for Ub footprints directly from trypsin-digested cell lysates using an anti-K-ԑ-GG antibodies specific for the GGremnant on ubiquitylated lysines (Udeshi et al, 2013). In fact, recent studies with such antibodies in rice discovered 1,376 possible ubiquitylated proteins and revealed strong changes in ubiquitylation after a three-hr exposure to flg22 or fungal chitin (Chen et al, 2018).…”