Michael Lückmann, Roxana-Maria Amarandi, Natalia Papargyri, Mette H. Jakobsen, Elisabeth Christiansen, Lars J. Jensen, Aurel Pui, Thue W. Schwartz, Mette M. Rosenkilde, Thomas M. Frimurer
The human cytomegalovirus-encoded G protein-coupled receptor US28 is a constitutively active receptor, which can recognize various chemokines. Despite the recent determination of its 2.9 Å crystal structure, potent and US28-specific tool compounds are still scarce. Here, we used structural information from a refined US28:VUF2274 complex for virtual screening of >12 million commercially available small molecule compounds. Using a combined receptor- and ligand-based approach, we tested 98 of the top 0.1% ranked compounds, revealing novel chemotypes as compared to the ~1.45 million known ligands in the ChEMBL database. Two compounds were confirmed as agonist and inverse agonist, respectively, in both IP accumulation and Ca2+mobilization assays. The screening setup presented in this work is computationally inexpensive and therefore particularly useful in an academic setting as it enables simultaneous testing in binding as well as in different functional assays and/or species without actual chemical synthesis.
The LC/MS analysis was carried out using Advion Expression® CMS.
A: Our group is interested in bacterial natural product chemistry, with projects ranging from the directed discovery of new natural products by genome mining and heterologous pathway expression, the elucidation of individual biocatalytic reactions by in vitro studies and the application of the respective biosynthetic enzymes in organic synthesis. This also involves significant synthetic work, for example to prepare enzyme substrates, validate natural product structure elucidation, or to realize chemo-enzymatic natural product total syntheses.
Q: WHAT WAS YOUR PREVIOUS WORK FLOW OR CHALLENGES?
A: While NMR spectroscopy is an analytical tool that is meanwhile accessible to chemical research practically at any time, making standard 1H NMR experiments available within minutes to few hours after a reaction work up, MS spectrometry can sometimes be challenging to conduct sufficiently fast to allow for an efficient work flow: MS equipment in academia is typically located in service units with relatively few machines for whole departments, often leading to waiting times of one to several days for an analysis. While MS would be the perfect tool to directly monitor the success of a chemical reaction in situ or to quickly evaluate the outcome of enzymatic transformations, above restrictions prevent this tool from being regularly and efficiently used.
Q: WHY DID YOU INCORPORATE THE EXPRESSION CMS INTO YOUR LABORATORY?
A: We integrated the expression CMS into our analytical equipment to obtain fast information on our synthetic and biocatalytic reactions, as well as on the content of whole bacterial raw extracts. While the machine is usually used for direct injections and for recording of MS spectra from TLC reaction controls using the Plate Express system, LC-MS measurements of enzymatic reaction set-ups and raw extracts are run overnight. To our surprise, even the detection of post-translational modifications on small proteins can accurately be detected with the CMS, providing us with an additional tool even for protein characterization. This now gives us fast initial MS data for all applications important to our lab, thus significantly streamlining our research work.
Q: WHO WOULD YOU RECOMMEND TO PURCHASE THE EXPRESSION CMS?
A: The expression CMS is the perfect MS system for any research laboratory involved in synthetic organic chemistry and chemical analytics of small molecules to proteins. The system is easy to use and maintain and can thus be used by many individuals. It is therefore particularly well-suited for academic research laboratories.
Shared live at the 254th ACS National Meeting in Washington, D.C. –
John Matson, Ph. D., Matson Research Group on Macromolecular and Supramolecular Chemistry, Assistant Professor, Virginia Tech.
Listen to the recorded workshop session highlighting the role of H2S in the human body and how compact mass spectrometry is used to assay peptides via direct injection for reaction monitoring, and coupled with HPLC for purification.
Advion Interchim Scientific’s range of AVANT®, high performance, liquid chromatography systems can be used standalone with UV and UV/Vis detector options, or with the expression® compact mass spectrometer to provide seamlessly integrated LC/CMS under the full control of Advion Interchim Scientific’s simple, intuitive Mass Express software suite.
Modular, stackable design, with many options, provides custom solutions for both HPLC and UHPLC needs. From the simplest manual injection HPLC to a fully automated, streamlined UHPLC system and everything in-between, the AVANT® series can be configured to fit your analytical requirements and your budget.
Cost effective, multidimensional GRAS (generally recognized as safe) counterfeit protection of drugs based on uniformly 13C labeled amino acids with compact mass spectrometry detection.
Quant Express, an addition to Advion’s user-friendly Mass Express 4.0 software suite, features quantitation to create a fully integrated software system for expression CMS users.
A: The research in the Biosyn group is focused on the design, synthesis and function of the four major types of biomolecules: nucleic acids, carbohydrates, peptides and lipids and hybrid structures thereof. These biomolecules and their derivatives are used in drug discovery and chemical biology, to develop synthetic methodology or as an inspiration for mimetic design.
Q: WHAT WAS YOUR PREVIOUS EXPERIENCE?
A: I started as organic chemist on Pt-adducts on self-made DNA fragments under supervision of Professor Dr. J. Reedijk. Following that, I switched to synthesis of bio-organic molecules on solid support under supervision of Professor Dr. J.van Boom. Currently I am working with sophisticated analytical equipment including many (prep) LCMS systems under supervision of Professor Dr. H. Overkleeft. With this equipment we show our facility to many other workgroups within and outside the University of Leiden.
Q: WHY DID YOU INCORPORATE THE EXPRESSION CMS INTO YOUR LABORATORY?
A: It’s easy, walk-up use with straightforward swapping of ESI and APCI sources make it a reliable, productive tool for determining synthetic success.
Q: HOW DID THE EXPRESSION CMS HELP RESOLVE YOUR CHALLENGES?
A: I incorporated the expression CMS as TLC/MS system because of its simplicity, low cost and the brilliant combination with the Plate Express (as a TLC interface).
Q: TO WHOM WOULD YOU RECOMMEND THE EXPRESSION CMS?
A: I recommend the expression CMS to people who want to use a robust system with simple handling (like organic chemists).
Q: WHAT IS THE FOCUS OF YOUR LAB’S RESEARCH? A: The synthesis of modified histidines for solid-phase peptide synthesis and new routes to exocyclic allenes.
Q: WHAT WAS YOUR PREVIOUS WORK FLOW OR CHALLENGE?
A: Some Nim-tritylated histidine analogues and exocyclic allenes provided weak or no molecular ions using other mass spectrometry methods.
Q: WHY DID YOU INCORPORATE THE EXPRESSION® CMS INTO YOUR LABORATORY?
A: It’s easy, walk-up use with straight-forward swapping of ESI and APCI sources make it a reliable, productive tool for determining synthetic success.
Q: HOW DID THE EXPRESSION® CMS HELP RESOLVE YOUR CHALLENGES?
A: ESI and APCI resolved extensive fragmentation observed with some analytes.
Q: TO WHOM WOULD YOU RECOMMEND THE EXPRESSION® CMS?
A: Organic synthesis labs and undergraduate programs needing a simple diagnostic tool for reaction outcomes.
Q: WHAT IMPRESSES YOU THE MOST ABOUT THE EXPRESSION® CMS?
A: Students find it straight-forward to use and its sources are easy to clean.
Published by the International Journal of Mass Spectrometry
Native liquid extraction surface analysis (LESA) mass spectrometry enables the direct sampling of protein complexes from a solid surface. We have previously demonstrated native LESA mass spectrometry of holomyoglobin (~17 kDa) from glass slides and tetrameric haemoglobin (~64 kDa) from dried blood spots and thin tissue sections. Here, we further explore the capabilities of this
emerging technique by investigating a range of proteins which exist in various oligomeric states in vivo.
Victor A.Mikhailov, Rian L.Griffiths, Helen J.Cooper,
Liquid Extraction Surface Analysis for Native Mass Spectrometry: Protein
Complexes and Ligand Binding, International Journal of Mass Spectrometry
http://dx.doi.org/10.1016/j.ijms.2016.09.011
