Mass Spectrometry Proteomics using TUBEs

Pan-selective Tandem Ubiquitin-Binding Entities (TUBEs) are engineered, high-affinity reagents composed of multiple ubiquitin-associated (UBA) domains that bind polyubiquitin chains with very high affinity. Their design enables pan-specific capture of all ubiquitin chain linkage types (K6, K11, K27, K29, K33, K48, K63, and M1)—overcoming the inherent bias of antibody-based methods like DiGly enrichment. Critically, TUBEs preserve native chain architecture during sample isolation by shielding polyubiquitinated proteins from deubiquitinating enzymes (DUBs) and proteasomal degradation. Compared to peptide-centric approaches, TUBEs streamline workflows from lysis to pulldown have the potential to reveal dynamic ubiquitin remodeling in cellular contexts—from proteasomal targeting signals (K11/K48) to inflammatory cascades (K63/M1 chains). These capabilities make TUBEs indispensable for identifying disease-linked ubiquitin signatures, such as K63-polyubiquitin accumulations in neurodegenerative aggregates or chain-ratio imbalances in therapy-resistant cancers.

While LifeSensors currently offers more up to date procedures specifically regarding our UM420: Ubiquitin Mass Spectrometry Kit, a complete kit designed for TUBE based pulldown and also offers a regularly used proteomics service for expert and experienced handling and support, TUBEs have also been used historically in other Mass Spec formats such as in this Nature Communication article. In this article TUBEs were utilized in a detailed procedure for whole proteomic analysis, see materials and methods. Common issues with low signal can be significantly enhanced by affinity enrichment via TUBEs, requiring less signal

MS proteomics enables untargeted discovery of proteins, post-translational modifications (PTMs), and interactomes in complex biological samples. Unlike antibody-based methods, it detects thousands of molecules simultaneously without prior target selection—revealing novel biomarkers, drug targets, and disease mechanisms.

LifeSensors has built on this concept by utilizing K48 and K63 specific TUBEs for targeted enrichment. These new TUBEs allow for a deeper more precise exploration of ubiquitination, by determining specifically what type of ubiquitination a set of samples may undergo, allowing for a specific new focus on these valuable drug discovery tools.