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Dualsystems-Biotech-Switzerland-LRC-TriCEPS-1

HATRIC-LRC for small molecules

Now available at Dualsystems

The TriCEPS technology platform has been further developed by the group of Professor Bernd Wollscheid from the ETH Zürich. The newest LRC platform technology is called HATRIC-LRC (HATRIC Ligand Receptor Capture) and enables to identify the targets of small molecule ligands at the cell membrane of living cells. Further, with the HATRIC platform less cells are needed for an experiment than with the original TriCEPS platform and N-, C-, and O-glycosylated targets can be identified.

The HATRIC-platform technology is based on a trifunctional cross linker called HATRIC. One arm of HATRIC contains an N-hydroxysuccinimid (NHS) to be able to bind to small molecules using a primary amine, the second arm contains a hydrazine to covalently bind the glycans of the unknown target proteins at the cell membrane and the third arm possesses an azide to enrich the target proteins.

In a first step the small molecule containing a primary amine is coupled to HATRIC on its first arm. Then the cells expressing the unknown targets and off-targets are mildly oxidized so that aldehydes form on the glycan moieties of the membrane associated proteins. Now, the second arm of HATRIC coupled to the small molecule is able to bind to the glycans at the cell surface. Once the small molecule binds its target the second arm of HATRIC covalently binds to the glycans of the unknown target proteins. In that state the cells expressing the unknown targets are still alive and the target molecules are in their natural microenvironment at the cell membrane. Due to the covalent link the situation is now fixed and the cells are lysed and the third arm of HATRIC is used to enrich the target proteins. The pulled down proteins are then digested with trypsin and identified and relative quantified by LC-MS/MS. Proteins that are enriched in one treatment arm compared to the other treatment arm are the target candidates.

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Customers Testimonials – LRC-TriCEPS Service

Testimonials from our customers who have used the LRC-TriCEPS technology – in collaboration with Dualsystems Biotech AG.

University of California San Francisco

"We have been trying to identify a receptor that has been sought after for nearly 30 years by laboratories across the globe. Thanks to Dualsystems service CaptiRec (LRC-TriCEPS), we now have a strong candidate receptor." indexDe'Broski R. Herbert Ph.D.
Assistant Professor in Residence
University of California San Francisco (UCSF)
2015-02-17T14:27:53+00:00
"We have been trying to identify a receptor that has been sought after for nearly 30 years by laboratories across the globe. Thanks to Dualsystems service CaptiRec (LRC-TriCEPS), we now have a strong candidate receptor." De'Broski R. Herbert Ph.D. Assistant Professor in Residence University of California San Francisco (UCSF)

Washington University School of Medicine

"Thanks to LRC-TriCEPS (CaptiRec) technology we were able to identify a long sought receptor of our ligand using genetically-engineered cell lines with the support of Dualsystems".Washington_University_in_StFumihiko Urano, MD, PhD
Samuel E. Schechter Professor of Medicine
Washington University School of Medicine
2015-02-17T14:30:12+00:00
"Thanks to LRC-TriCEPS (CaptiRec) technology we were able to identify a long sought receptor of our ligand using genetically-engineered cell lines with the support of Dualsystems".Fumihiko Urano, MD, PhD Samuel E. Schechter Professor of Medicine Washington University School of Medicine

Centro de Estudos de Doenças Crónicas

New publication in Nature Communications using the LRC-TriCEPS technology in collaboration with Alisson Gontijo
« The fruitful collaboration with Dualsystems Biotech using the LRC-TriCEPS (CaptiRec) technology showed that even on insect cells receptors could be identified »

Cedoc-logoAlisson M. Gontijo,
Principal Investigator at CEDOC
Centro de Estudos de Doenças Crónicas
2015-02-24T07:32:40+00:00
New publication in Nature Communications using the LRC-TriCEPS technology in collaboration with Alisson Gontijo « The fruitful collaboration with Dualsystems Biotech using the LRC-TriCEPS (CaptiRec) technology showed that even on insect cells receptors could be identified » Alisson M. Gontijo, Principal Investigator at CEDOC Centro de Estudos de Doenças Crónicas

Igenica Biotherapeutics

«Leveraging Dualsystems Biotech novel linker technology (LRC-TriCEPS) and its analytical data processing capabilities we were able to identify the heterophilic receptor for a highly-pursued immuno-oncology target.»Igenica-Biotherapeutics-logoDr. Edward van der Horst,
Senior Director, Preclinical Development
Igenica Biotherapeutics
2016-05-09T11:13:08+00:00
«Leveraging Dualsystems Biotech novel linker technology (LRC-TriCEPS) and its analytical data processing capabilities we were able to identify the heterophilic receptor for a highly-pursued immuno-oncology target.»Dr. Edward van der Horst, Senior Director, Preclinical Development Igenica Biotherapeutics

East Tennessee State University

For the ligand sample two unique receptors were identified. Flow cytometry analysis with siRNA induced knockdown of these proteins confirmed that the presence of the protein is needed for CTRP3 binding to occur. CONCLUSION The LRC-TriCEPS methodology was successful in identifying the receptor for CTRP3.Logo East Tennessee State UniversityJonathan M Peterson
Assistant Professor
East Tennessee State University
2016-05-30T15:20:03+00:00
“For the ligand sample two unique receptors were identified. Flow cytometry analysis with siRNA induced knockdown of these proteins confirmed that the presence of the protein is needed for CTRP3 binding to occur. CONCLUSION The LRC-TriCEPS methodology was successful in identifying the receptor for CTRP3.”Jonathan M Peterson Assistant Professor East Tennessee State University

Medizinische Hochschule Hannover

With the support of Dualsystems Biotech and their LRC-TriCEPS technology we were able to identify receptor candidates for our peptide with renoprotective properties in kidney injury.

mh-hannover-logoDr. rer. nat. Inga Sörensen-Zender
Postdoc
Medizinische Hochschule Hannover

 
2016-09-26T10:26:51+00:00
“With the support of Dualsystems Biotech and their LRC-TriCEPS technology we were able to identify receptor candidates for our peptide with renoprotective properties in kidney injury.” Dr. rer. nat. Inga Sörensen-Zender Postdoc Medizinische Hochschule Hannover  

The Rockefeller University

"We are very pleased with the work product of Dualsystems Biotech. The TriCEPS reagent allowed us to identify a novel ligand-extracellular matrix protein receptor interaction, which was not possible using traditional techniques. The Dualsystems team were very helpful in tailoring the experimental conditions to fit our biological question."rockefeller-university-logoManish Ponda, M.D., M.S.
Assistant Professor of Clinical Investigation
The Rockefeller University
2016-10-10T14:21:51+00:00
"We are very pleased with the work product of Dualsystems Biotech. The TriCEPS reagent allowed us to identify a novel ligand-extracellular matrix protein receptor interaction, which was not possible using traditional techniques. The Dualsystems team were very helpful in tailoring the experimental conditions to fit our biological question."Manish Ponda, M.D., M.S. Assistant Professor of Clinical Investigation The Rockefeller University

University of Manitoba

"Every aspect of our experience with Dualsystems Biotech was outstanding.  They provided excellent customer service and technical support throughout the entire process.  Their LRC-TriCEPS technology allowed us to identify several new candidate receptors for our protein of interest in rat primary neurons, and this has created new and exciting avenues for our research."

University of Manitba-logoSari S. Hannila, PhD
Associate Professor, Department of Human Anatomy and Cell Science
Associate Member, Spinal Cord Research Centre
Max Rady College of Medicine, Rady Faculty of Health Sciences
University of Manitoba
2017-06-12T10:05:43+00:00
"Every aspect of our experience with Dualsystems Biotech was outstanding.  They provided excellent customer service and technical support throughout the entire process.  Their LRC-TriCEPS technology allowed us to identify several new candidate receptors for our protein of interest in rat primary neurons, and this has created new and exciting avenues for our research." Sari S. Hannila, PhD Associate Professor, Department of Human Anatomy and Cell Science Associate Member, Spinal Cord Research Centre Max Rady College of Medicine, Rady Faculty of Health Sciences University of Manitoba

Münster University Hospital (UKM)

"Dualsystems Biotech’s approach to receptor identification with the TriCEPS reagent gave us a powerful platform with great support for a successful and fast data generation, when conventional approaches had previously failed."Münster University Hospital (UKM) Dr. rer. nat. Martin Herold
Working group from Prof. Dr. med. Luisa Klotz
Münster University Hospital (UKM), Germany
2017-08-24T14:48:20+00:00
"Dualsystems Biotech’s approach to receptor identification with the TriCEPS reagent gave us a powerful platform with great support for a successful and fast data generation, when conventional approaches had previously failed."Dr. rer. nat. Martin Herold Working group from Prof. Dr. med. Luisa Klotz Münster University Hospital (UKM), Germany

University of Miami, Miller School of Medicine

We are very pleased with the experience and interaction on very high professional level with Dualsystems Biotech. As a result of such collaboration the new receptor MUC5B was identified in human chondrosarcoma cells for the first time for antiproliferative neuropeptide PRP-1. Read more
I would like to thank once again the company and, particularly, Dr Helbling for his attention and collaboration.
Dualsystems-Logo-University of Miami, Miller School of MedicineDr Karina Galoian
Research associate professor
University of Miami, Miller School of Medicine
Department of Orthopedic surgery
Miami, Florida, USA
2017-11-16T08:07:04+00:00
We are very pleased with the experience and interaction on very high professional level with Dualsystems Biotech. As a result of such collaboration the new receptor MUC5B was identified in human chondrosarcoma cells for the first time for antiproliferative neuropeptide PRP-1. Read more I would like to thank once again the company and, particularly, Dr Helbling for his attention and collaboration. Dr Karina Galoian Research associate professor University of Miami, Miller School of Medicine Department of Orthopedic surgery Miami, Florida, USA

QIMR Berghofer Medical Research Institute

“With the help of the team at Dualsystems we confirmed a potential receptor which has lead to a Nature paper submission and successful funding of a research grant. The LRC-TriCEPS experiment was straightforward and the process was cost effective. We look forward to using TriCEPS in the future.”QIMR Berghofer Medical Research Institute LogoAnita Burgess  |  PhD
Hepatic Fibrosis Group
QIMR Berghofer Medical Research Institute, Australia
2017-12-12T13:21:33+00:00
“With the help of the team at Dualsystems we confirmed a potential receptor which has lead to a Nature paper submission and successful funding of a research grant. The LRC-TriCEPS experiment was straightforward and the process was cost effective. We look forward to using TriCEPS in the future.”Anita Burgess  |  PhD Hepatic Fibrosis Group QIMR Berghofer Medical Research Institute, Australia

Biomedical Research Institute

PUBLICATION:  Li Z, Zeppa JJ, Hancock MA, McCormick JK, Doherty TM, Hendy GN, and Madrenas J.  Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells. J Immunol. 2018; 200: 1471-1479.

The identification of a T cell co-receptor for staphylococcal superantigens had been challenging due to the structural features of the interaction and its kinetics.  However, working with Dualstystems Biotech AG, and with Dr. Paul Helbling in particular, and using the LRC-TriCEPS technology, we were able to identify a candidate that was subsequently corroborated by biochemical and functional assays.   We are very happy with this collaboration , and sincerely recommend it for the identification of novel receptor or co-receptor candidates.
(Quim) Madrenas, MD, PhD, FCAHS
Chief Scientific Officer
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
Torrance, USA
2018-04-19T11:18:18+00:00
PUBLICATION:  Li Z, Zeppa JJ, Hancock MA, McCormick JK, Doherty TM, Hendy GN, and Madrenas J.  Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells. J Immunol. 2018; 200: 1471-1479. The identification of a T cell co-receptor for staphylococcal superantigens had been challenging due to the structural features of the interaction and its kinetics.  However, working with Dualstystems Biotech AG, and with Dr. Paul Helbling in particular, and using the LRC-TriCEPS technology, we were able to identify a candidate that was subsequently corroborated by biochemical and functional assays.   We are very happy with this collaboration , and sincerely recommend it for the identification of novel receptor or co-receptor candidates. (Quim) Madrenas, MD, PhD, FCAHS Chief Scientific Officer Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center Torrance, USA

LRC-TriCEPS customers worldwide

Over 200 satisfied customers from 28 countries.

5+

Years working with LRC-TriCEPS

200+

LRC-TriCEPS clients served

50+

Cell types used for LRC-TriCEPS

18+

Years of experience

LRC-TriCEPS publications worldwide

LRC-TriCEPS / HATRIC-LRC Publications

Concerning the LRC-TriCEPS or HATRIC-LRC platforms.

Leukocyte differentiation by histidine-rich glycoprotein/stanniocalcin-2 complex regulates murine glioma growth through modulation of anti-tumor immunity

Reference
Francis P RocheIlkka PietiläHiroshi KaitoElisabet O SjöströmNadine SobotzkiOriol NoguerTor Persson SkareMagnus EssandBernd WollscheidMichael Welsh and Lena Claesson-Welsh
Received January 27, 2018, Revision received April 21, 2018, Accepted June 19, 2018, Copyright ©2018, American Association for Cancer Research. PDF
Abstract

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and anti-tumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and co-localization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG treated gliomas displayed decreased numbers of Interleukin-35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control anti-tumor immunity.

Author

Francis P Roche1Ilkka Pietilä2Hiroshi Kaito3Elisabet O Sjöström1Nadine Sobotzki4Oriol Noguer1Tor Persson Skare1Magnus Essand1Bernd Wollscheid5Michael Welsh2, and Lena Claesson-Welsh1,*


  1. 1Department of Immunology, Uppsala University

  2. 2Department of Medical Cell Biology, Uppsala University

  3. 3Department of Immunology, Uppsala university

  4. 4Department of Health Sciences, ETH Zurich

  5. 5Department of Health Sciences, ETH Zürich
  1. * Corresponding Author:

Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions

Reference
Lisa Lasswitz, Naresh Chandra, Niklas Arnberg, Gisa Gerold jmb Journal of Molecular Biology, doi.org/10.1016/j.jmb.2018.04.039 Received 15 February 2018, Revised 24 April 2018, Accepted 30 April 2018, Available online 7 May 2018.
Abstract

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.

Keywords

adenovirus
virus entry
proteomics
glycomis
host cell interactions

Abbreviations

HAdVs

human adenoviruses

LSFs

long-shafted fibers

SSFs

short-shafted fibers

SA

Sialic acid

IAVs

influenza A viruses

GAGs

glycosaminoglycans

HS

heparan sulfate

HSPG

heparan sulfate proteoglycan

HBGAs

histo blood group antigens

GSLs

glycosphingolipids

CAR

coxsackie and adenovirus receptor

VCAM-1

vascular cell adhesion molecule-1

MHC-I

major histocompatibility complex 1

CFG

Consortium for Functional Glycomics

SG

shotgun glycomics

ICL

Imperial College of London

VOPBAs

virus overlay protein binding assays

VAPs

virus attachment proteins

AE-MS

affinity enrichment mass spectrometry
Author
Lisa Lasswitz1Naresh Chandra2,3Niklas Arnberg2,3Gisa Gerold1,2,4
Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, 30625 Hannover, Germany
Department of Clinical Microbiology, Virology, Umeå University, SE-90185 Umeå, Sweden
Molecular Infection Medicine Sweden (MIMS), Umeå University, SE-90185 Umea, Sweden
Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, SE-90185 Umea, Sweden

HATRIC-based identification of receptors for orphan ligands

Reference
Nadine Sobotzki, Michael A. Schafroth, Alina Rudnicka, Anika Koetemann, Florian Marty, Sandra Goetze, Yohei Yamauchi, Erick M. Carreira & Bernd Wollscheid Nature Communications, volume 9, Article number: 1519 (2018) doi:10.1038/s41467-018-03936-z Published online: 
Abstract

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu.

Author

Nadine Sobotzki, Michael A. Schafroth, Alina Rudnicka, Anika Koetemann, Florian Marty, Sandra Goetze, Yohei Yamauchi, Erick M. Carreira & Bernd Wollscheid

Staphylococcal Superantigens Use LAMA2 as a Coreceptor GPCT signaling To Activate T Cells

Reference
Zhigang Li, Joseph J. Zeppa, Mark A. Hancock, John K. McCormick, Terence M. Doherty, Geoffrey N. Hendy and Joaquín Madrenas J Immunol January 15, 2018, ji1701212; DOI: https://doi.org/10.4049/jimmunol.1701212  (Published online February 5, 2018) This work was supported by the Canadian Institutes for Health Research. J.M. holds a tier I Canada Research Chair in Human Immunology. The Department of Microbiology and Immunology Flow Cytometry and Cell Sorting Facility and McGill Surface Plasmon Resonance–Mass Spectrometry Facility are supported by the Canada Foundation for Innovation.
Abstract

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein–coupled receptor (GPCR) leading to PLCβ activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.

Author

Zhigang Li, Joseph J. Zeppa, Mark A. Hancock, John K. McCormick, Terence M. Doherty, Geoffrey N. Hendy and Joaquín Madrenas

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

Reference
International Journal of Oncology, published online on: November 9, 2017   doi.org/10.3892/ijo.2017.4199 Authors: Karina Galoian, Silva Abrahamyan, Gor Chailyan, Amir Qureshi, Parthik Patel, Gil Metser, Alexandra Moran, Inesa Sahakyan, Narine Tumasyan, Albert Lee, Tigran Davtyan, Samvel Chailyan and Armen Galoyan Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed.
Abstract

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

Author

Karina Galoian, Silva Abrahamyan, Gor Chailyan, Amir Qureshi, Parthik Patel, Gil Metser, Alexandra Moran, Inesa Sahakyan, Narine Tumasyan, Albert Lee, Tigran Davtyan, Samvel Chailyan and Armen Galoyan

Phenotypic screening—the fast track to novel antibody discovery

Reference
ScienceDirekt, doi.org/10.1016/j.ddtec.2017.03.004 Department of Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge CB21 6GH, UK Available online 25 April 2017
Abstract

The majority of antibody therapeutics have been isolated from target-led drug discovery, where many years of target research preceded drug program initiation. However, as the search for validated targets becomes more challenging and target space becomes increasingly competitive, alternative strategies, such as phenotypic drug discovery, are gaining favour. This review highlights successful examples of antibody phenotypic screens that have led to clinical drug candidates. We also review the requirements for performing an effective antibody phenotypic screen, including antibody enrichment and target identification strategies. Finally, the future impact of phenotypic drug discovery on antibody drug pipelines will be discussed.

Author

Section editors: Neil O Carragher – Institute of Genetics and Molecular Medicine, Cancer Research UK Edinburgh Centre, University of Edinburgh, Edinburgh, United Kingdom. Jonathan A. Lee – Quantitative Biology, Eli Lilly and Company, Indianapolis, Indiana. Ellen L. Berg – BioMAP Systems Division of DiscoverX, DiscoverX Corporation.

Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology

Reference
PLoS One. 2016 Oct 11;11(10):e0164593. doi: 10.1371/journal.pone.0164593. eCollection 2016. Li Y1, Ozment T2, Wright GL1, Peterson JM1,3. We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and>INS as a control ligand was performed on the H4IIE rat hepatoma cell line.
Abstract

Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the>INS receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells.

Conclusion

The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3.

Relevance

The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

Author

Li Y1, Ozment T2, Wright GL1, Peterson JM1,3.

  • 1Quillen College of Medicine, Department of Biomedical Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.
  • 2Quillen College of Medicine, Department of Internal Medicine, East Tennessee State University, Johnson City, Tennessee, United States of America.
  • 3College of Public Health, Department of Health Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.

Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5

Reference
Current Issue – vol. 113 no. 45 – Manish P. Ponda,  E7059–E7068, doi: 10.1073/pnas.1615671113 Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan) Manish P. Pondaa and Jan L. Breslowa,1 Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. To identify potential receptors in an unbiased manner, we used ligand–receptor capture (LRC-TriCEPS) technology as a tool for detecting T-lymphocyte surface proteins that physically interact with H497–K520 (Fig. 5A) (20). Transferrin was used as a control ligand to eliminate nonspecific interactions with the TriCEPS reagent.
Abstract

TriCEPS Ligand-Receptor Capture.

The TriCEPS reagent is a proprietary trifunctional molecule with three key moieties: (i) an amine-reactive group capable of binding a peptide ligand of interest, (ii) a cross-linking group capable of bonding to oxidized glycans, and (iii) an affinity tag for downstream extraction (20, 50). These studies were performed by DualSystems Biotech. Briefly, either human H497–K520 or transferrin was coupled to the TriCEPS reagent. The TriCEPS–ligand complex was added to CCRF-CEM cells in complete medium in the presence or absence of zinc (100 μM), after treatment with an oxidizing reagent to oxidize surface glycoproteins. Cells were then lysed and processed as described, including LC-MS/MS analysis of captured peptides (20). Comparisons between conditions were made in triplicate to determine the relative fold enrichment of a given protein. P values were determined for pairwise comparisons and adjusted for multiple comparisons.

Author

Manish P. Ponda

aLaboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065

Jan L. Breslow

aLaboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization

Reference
Abstract

Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41’s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue–NP41-bound nerves, followed by hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10−7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve “ghosts” months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.

Author

Heather L. Glasgow

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Michael A. Whitney

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Larry A. Gross

bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;

Beth Friedman

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Stephen R. Adams

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Jessica L. Crisp

bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;

Timon Hussain

cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;

Andreas P. Frei

dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Karel Novy

dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Bernd Wollscheid

dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Quyen T. Nguyen

cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;

Roger Y. Tsien

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;

eDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093

Identification of cell surface receptors for the novel adipokine CTRP3

Reference
April 2016, The FASEB Journal, vol. 30 no. 1 Supplement 1249.2 Jonathan M Peterson C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects throughout the body. Our initial work shows promise in the development of CTRP3-induced cellular processes as a means to combat nonalcoholic fatty liver disease (NAFLD). Clinically, NAFLD is defined as the excessive accumulation of fat in the liver, usually due to obesity, and NAFLD effects 1 in 10 Americans. Our previous data show that when high fat fed mice are treated with CTRP3 they are protected from developing NAFLD. However, the mechanism for this effect remains unclear. The purpose of this project was to identify the unknown receptor that mediates the hepatic actions of CTRP3. .
Abstract

Methods

We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and INS as the control ligand was performed on H4IIE rat hepatoma cell line. Additional analysis using siRNA induced knockdown of the identified receptors was used to validate the findings.

Results

Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS to the cell surface glycans. In total, an enrichment of glycopeptides of 11% (261 peptides) was observed. Under these conditions, INSR (Insulin receptor) could be identified and quantified in the control dataset. For the ligand sample two unique receptors were identified. Flow cytometry analysis with siRNA induced knockdown of these proteins confirmed that the presence of the protein is needed for CTRP3 binding to occur.

Conclusion

The LRC-TriCEPS methodology was successful in identifying the receptor for CTRP3.

Relevance

The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

Author

Jonathan M Peterson

Health Sciences, East Tennessee State University, Johnson City, TN

This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing

Reference
Nature Communications 6, Article number: 8732 (2015), doi:10.1038/ncomms9732
Received:
Accepted:
Published online:
Andres Garelli, Fabiana Heredia, Andreia P. Casimiro, Andre Macedo, Catarina Nunes, Marcia Garcez, Angela R. Mantas Dias, Yanel A. Volonte, Thomas Uhlmann, Esther Caparros, Takashi Koyama & Alisson M. Gontijo
Abstract

How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in INS-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.

Author

A Mass Spectrometric-Derived Cell Surface Protein Atlas

Reference
Published: April 20, 2015 – http://dx.doi.org/10.1371/journal.pone.0121314 Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa).
Abstract

The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

Author

Damaris Bausch-Fluck

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland

Andreas Hofmann

Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Thomas Bock

Current Address: European Molecular Biology Laboratory, Heidelberg, Germany

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Andreas P. Frei

Current Address: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, United States of America

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Ferdinando Cerciello

Current Address: James Thoracic Center, James Cancer Center, The Ohio State University Medical Center, Columbus, Ohio, United States of America

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Laboratory of Molecular Oncology, University Hospital Zurich, Zurich, Switzerland

Andrea Jacobs

Current Address: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Hansjoerg Moest

Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Ulrich Omasits

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland

Rebekah L. Gundry

Affiliation Department of Biochemistry, Medical College of Wisconsin, Wisconsin, Milwaukee, United States of America

Charles Yoon

Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada

Ralph Schiess

Current Address: ProteoMediX AG, Schlieren, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Alexander Schmidt

Current Address: Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Paulina Mirkowska

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland

Anetta Härtlová

Current Address: College of Life Sciences, University of Dundee, Dundee, United Kingdom

Affiliation Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic

Jennifer E. Van Eyk

Current Address: Cedars-Sinai, Clinical Biosystem Research Institute, Los Angeles, California, United States of America

Affiliation Department of Medicine, Biological Chemistry and Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America

Jean-Pierre Bourquin

Affiliation Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland

Ruedi Aebersold

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland, Faculty of Science, University of Zurich, Zurich, Switzerland

Kenneth R. Boheler

Affiliations SCRMC, LKS Faculty of Medicine, Hong Kong University, Hong Kong, Hong Kong SAR, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America

Peter Zandstra

Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada

Bernd Wollscheid

* E-mail: wbernd@ethz.ch

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland

Competing Interests

The authors have declared that no competing interests exist.

Author Contributions

Conceived and designed the experiments: DBF BW KRB PZ RA. Performed the experiments: DBF AH TB APF FC AJ HM RLG CY RS AS PM AH. Analyzed the data: DBF UO. Contributed reagents/materials/analysis tools: AS APF FC TB BW. Wrote the paper: DBF BW. Supervised experiments: JVE JPB.

Protter

Intro
Protter — the open-source tool for visualization of proteoforms and interactive integration of annotated and predicted sequence features together with experimental proteomic evidence. The ability to integrate and visualize experimental proteomic evidence in the context of rich protein feature annotations represents an unmet need of the proteomics community. Protter, a web-based tool that supports interactive protein data analysis and hypothesis generation by visualizing both annotated sequence features and experimental proteomic data in the context of protein topology. Protter supports numerous proteomic file formats and automatically integrates a variety of reference protein annotation sources, which can be readily extended via modular plug-ins. A built-in export function produces publication-quality customized protein illustrations, also for large datasets. Visualizations of surfaceome datasets show the specific utility of Protter both for the integrated visual analysis of membrane proteins and peptide selection for targeted proteomics.
Abstract

Protter-Tool

Author

Ligand-based receptor identification on living cells and tissues using TRICEPS

Reference
Affiliations ¦ Contributions ¦Corresponding authors Nature Protocols 8, 1321–1336 (2013) doi:10.1038/nprot.2013.072
Published online 13 June 2013
Abstract

Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS—one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., INS) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.

Author

Flex your TRICEPS

Intro
Nature Chemical Biology 8, 950 (2012) doi:10.1038/nchembio.1126 Published online 26 November 2012 Identifying ligands for receptors in live cells can provide valuable information, providing insight into signaling pathways as well as drug targets, but these interactions can be difficult to detect and quantify. Frei et al. now report a trifunctional chemoproteomic reagent called TRICEPS that binds glycosylated receptors on live cells and allows for the purification of the ligand-receptor complex and identification of receptor-derived peptides by MS. TRICEPS contains three functional groups: an N-hydroxysuccinimide ester for ligand conjugation, a protein….
Abstract
Author