TUBE-based Mass Spectrometry Proteomics
Tandem Ubiquitin Binding Entities (TUBEs) are powerful reagents for enrichment of polyubiquitylated proteins. At LifeSensors, we have used this remarkable technology of TUBEs with our mass spectrometry expertise to provide the customer with a quick and easy way to perform both qualitative and quantitative proteomics. As a LifeSensors customer, your proteomics project begins when our team receives your sample as a frozen cell pellet. Our required size is a minimum of 2.5x107 cells / 5 mg protein. We recommend a pellet size of, 5-10x107 cells / 10-20 mg protein. Once your sample is received, we perform cell lysis, TUBE enrichment of polyubiquitylated proteins, in-gel trypsin digestion of enriched sample, and LC-MS/MS analysis.
Upon completion of the analysis, you will receive a list of peptides and corresponding proteins that have been identified. This information will help you publish your results or plan your next assay.
Each experiment is unique, therefore the time required for individual experiments may differ. However, we do provide a rough estimate based upon the number of samples you want analyzed:
3-5 weeks turnaround for 1-4 samples
4-6 weeks for 5-9 samples
5-7 weeks for 10-15 samples
Please contact us for time estimates for analysis of 16 or more samples.
TUBE proteomics gives you a global understanding of the proteins that are ubiquitylated in each of your samples. Accordingly, you can discover changes in the ubiquitylation profile of your cell or tissue samples in response to different treatments, including siRNA knockdown, stress induction, or drug treatment. We will provide general guidance to facilitate your analysis of the raw data. TUBE proteomics will allow the discovery of novel ubiquitylation substrates. This methodology can be applied to discover new biological regulation pathways, monitor the cellular effects of a novel drug, or identify prospective disease biomarkers.
TUBE-based enrichment of polyubiquitylated proteins has proven key for the progress of ubiquitin proteomics. LifeSensors has developed the proprietary technology to identify cellular proteins that are ubiquitylated using TUBE-based proteomics. A brief overview of this technology is depicted in the following figure:
Workflow schematic of TUBE proteomics. Treated cells are enriched using TUBEs (TUBE1 and K48). The enriched polyubiquitylated proteins are pixelated by SDS-PAGE and digested with trypsin. Individual ubiquitylated proteins are identified by the presence of at least 2 peptides in at least 2 of 3 replicates. Ub linkage types are identified by K-ε-GG peptides corresponding to covalent Ub-Ub modification at a particular lysine.
LifeSensors’ pan (TUBE1 and TUBE2) as well as linkage-selective (M1-, K48-, and K63- specific) TUBEs provide isolation of respective polyubiquitylated proteins. The TUBE enrichment kit contains solutions to provide simple procedure to lyse cells, wash the non-specific binding proteins, and elute proteins in solution compatible for further protease digestion. TUBE proteomics services include following four steps:
TUBE Enrichment Kit - Kit for enrichment of polyubiquitinated protein: Magnetic-TUBE1 and TUBE2 beads, Cell lysis buffer, Wash buffer and Elution buffer
K48 TUBE Enrichment Kit - Kit for enrichment of K48-polyubiquitinated proteins: Magnetic K48-linkage selective TUBE, Cell lysis buffer, Wash buffer and Elution buffer
K63 TUBE Enrichment Kit - Kit for enrichment of K63-polyubiquitinated proteins: Magnetic K63-linkage selective TUBE, Cell lysis buffer, Wash buffer and Elution buffer
We performed pan-selective TUBE and K48 TUBE HF pulldowns of SH-SY5Y cells. We analyzed the peptides isolated for ubiquitin K-ε-GG remnants to confirm the linkage specificity of the isolation. From the Pan-selective TUBE, we confirm that we can pulldown differently-linked ubiquitylated proteins. Moreover, we showed that K48 TUBE HF performs extremely well, with 92% fidelity for K48 linkage type.
Linkage distribution of polyubiquitin chains based on LC-MS/MS identification of ubiquitin remnant peptides. Ubiquitylated proteins were enriched from cell lysates using Magnetic TUBE1 (left), and K48 TUBE HF Biotin (right). Ub linkage types were identified by K-ε-GG peptides corresponding to covalent Ub-Ub modification at a particular lysine.
Published data using TUBE-based Mass Spectrometry
Silva et al.1 have used linkage-specific TUBE-based Mass Spectrometry to identify >100 novel K63 polyubiquitylated targets that were significantly enriched in ribosomal proteins. Using this and other data, they show that oxidative stress response is modulated by K63-linked polyubiquitylation.
Ribosomal proteins are a main target of K63 polyubiquitylation under H2O2 stress A. Experimental setup for the analysis of K63-linked ubiquitylated proteins using SILAC-based quantitative mass spectrometry. B. Anti-K63 ubiquitin western blot of lysates from WT and K63R mutant cells, C. Chart showing gene ontology (GO) annotation for K63 ubiquitinated targets using DAVID functional annotation tool. (*) GO enrichment significant at FDR < 0.005 %.\
In 2016, Mata-Cantero et al.2 used TUBE-based mass-spectrometry to identify major components of the ubiquitin proteome of both Plasmodium falciparum and its host during different life stages.
Identification of ubiquitylated proteins using TUBEs-LC-MS/MS method. Intraerythrocytic Developmental Cycle of P. falciparum is shown. Synchronized P. falciparum iRBC at 40% parasitaemia from rings, trophozoites and schizonts stages were collected and frozen. TUBE enriched proteins from iRBC at different stages and uRBC were captured using TUBEs or GST (control) previously crosslinked with DMP to agarose beads. After exhaustive washes, proteins captured were eluted, cleaned by precipitation and resolved by electrophoresis (PAGE). Bands with proteins were analyzed by LC-MS/MS.
1. Silva et al. K63 polyubiquitination is a new modulator of the oxidative stress response Nature Structural Molecular Biology 22 (2015) 116–123
2. L. Mata-Cantero et al., New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach, Journal of Proteomics, 139 (2016) 45–59