Post translational modification (PTM) of proteins plays a fundamental role in their function. Attachment of ubiquitin to epsilon (ε) amino group of lysine in target proteins represent the major PTM. In addition to the attachment of ubiquitin (ubiquitylation) to lysines, other ubiquitin-like-proteins (UBLs) such as SUMO (sumoylation), NEDD8 (neddylation) and ISG15 (isgynylation) are equally well represented PTMs of proteins. Ubiquitylation of proteins is a hallmark of proteins, provides clues to many diseases, and acts as a biomarker. Modern drug discovery and personalized medicine relies heavily upon proteomics to identify these signatures. A variety of methods are available to detect ubiquitin or UBL PMTs of proteins. We encourage you to contact our senior staff to learn about how LifeSensors’ PTM technology can help in your research and drug discovery process. One of the best ways to perform ubiquitin and UBL proteomics is to enrich ubiquitylated proteins with LifeSensors’ TUBE technologies and perform mass spectrometry studies. Lifesensors offers state of the art services to determine sites of ubiquitylation and UBLs by utilizing a combination of TUBE and UbiSight antibody applications. The combination of these two powerful technologies enables researchers to unravel the proteome and ubiquitome.
TUBE Based Mass Spectrometry Proteomics
Mass Spec Proteomics: Traditional methods for mass spectrometry based proteomics, such as SILAC (stable isotope labeling with amino acids in cell culture) and others, are insensitive, not reproducible, and costly and time consuming. LifeSensors’ affinity matrices bind to all poly-ubiquitin chains with 1-10 nM affinity. This has solved major problems common to mass spectrometry proteomics. LifeSensors has developed K63-, K48-, and M1(linear)-chain selective TUBEs. LifeSensors is actively engaged in developing K6, K11, K29, K27, and K33 polyubiquitin chain-selective TUBEs. The combination of the TUBE-based affinity technology as well as targeted mass spectrometry is the most powerful way to detect the alternations in PMTs and identify signatures for research and biomarkers. LifeSensors’ ubiquitin proteomics technology can detect ultralow levels of ubiquitylated biomarkers from tissues and cells. LifeSensors provides proteomics services that combines TUBE technology with Tandem Mass and synchronous precursor selection-based MS3 (SPS-MS3) mass spectrometry to establish and validate your biomarkers in cell and tissue samples. LifeSensors’ proteomics service is specially designed to monitor biomarkers for drugs that target ubiquitin proteasome system enzymes in clinical trials.
UbiSight Proteomics
UBiSight Proteomics: A common feature of ubiquitin and ubiquitin-like-protein (UBL) modifications is the C-terminus (Arg-Gly-Gly) of ubiquitin and UBLs, which is conjugated to a lysine residue of the target protein. Standard mass spectrometry methods utilize trypsin digestion, which leaves a di-glycine “remnant” attached to the lysine of target protein peptide (Gly-Gly-ε-Lys or diGly). Although ubiquitin proteomics is well established and numerous papers make use of the di-Gly-ε- lys antibody from Cell Signaling Technologies. The data suggest that the Gly-Gly-ε-Lys antibody-based mass spectrometry is plagued with many problems. For instance, (1) Ubiquitylated peptides are not quantitatively precipitated by the antibody. (2) Large amount of total starting material (protein) is required to monitor ubiquitin signatures from cells or tissue. (3) Compared to TUBE affinity technology that enriches ubiquitylated proteins, the cost of antibody per experiment is too high. (4) Lastly the di-Gly antibody method cannot differentiate between the C-terminal remnants of ubiquitin from other UBLs.
LifeSensors TUBE-based affinity technology enriches poly-ubiquitylated proteins, which overcomes many of the flaws in the di-Gly antibody-based mass spectrometry proteomics method. For the first time in proteomics, a combination of TUBE and UbiSight technologies can identify and distinguish between ubiquitin and UBLs conjugation sites at the proteome level. To accomplish this, we are developing a novel antibody that recognize the last 13 amino acids of ubiquitin attached to ε-Lys of target proteins. This technology is called UBiSight. UbiSight is a combination of TUBE-based affinity enriched poly-ubiquitylated proteins and the novel UBiSight antibody-based technology. This enables researchers to identify ubiquitylated proteins and substrates in a quantitative fashion, does not require any stable isotope labeling, and is highly efficient in identifying many novel PTM sites in the proteome. We invite you to talk to our team of experts who are pioneering the research of ubiquitin proteomics. LifeSensors can help you uncover your ubiquitin substrates, discover novel PTM biomarkers, analyze the effect of drugs on target tissues, and illuminate the landscape of the ubiquitin proteome with state-of-the-art technologies. This technology is highly quantitative, does not require SILAC (stable isotope labeling with amino acids in cell culture) and the di-Gly antibody. We are now poised to profile the genome-wide proteome of ubiquitin and ubiquitin-like protein specific conjugates. This will be the first research tool to distinguish between ubiquitin and UBL modification on a proteome level and will thus provide the first true mapping of the ubiquitin-specific landscape in cells. Should you have any questions about how our TUBE-based mass spectrometry or UbiSight assays can further your research, please contact our scientific team.
Microarrays for Ubiquitomics
The first stage in any drug discovery process is to search for a potential therapeutic target. For this reason, LifeSensors has developed a powerful and versatile protein microarray. This microarray enables researchers to identify both substrates for deubiquitylating enzymes (DUBs) and E3 ligase. In addition, LifeSensors makes use of its ubiquitin technologies to inform researchers if their specified substrates are being ubiquitylated or deubiquitylated. To learn more about this powerful technology, we invite you to contact us