scholarly journals Cleavable linkers and their application in MS-based target identification

2021 ◽  
Author(s):  
Hester A. Beard ◽  
Dimitris Korovesis ◽  
Suyuan Chen ◽  
Steven H. L. Verhelst
Keyword(s):  
Mass Spectrometry ◽  
Target Identification ◽  
Chemical Proteomics

In chemical proteomics workflows, cleavable linkers are increasingly used to facilitate target identification by mass spectrometry. This review discusses the various types of cleavable linkers and their application areas.

scholarly journals Development of a target identification approach using native mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miaomiao Liu ◽  
Wesley C. Van Voorhis ◽  
Ronald J. Quinn
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Target Identification ◽  
Native Mass Spectrometry ◽  
Pharmaceutical Drugs ◽  
Ligand Complex ◽  
Experimental Conditions ◽  
Protein Mixture ◽  
Native Ms ◽  
Drug Target Identification

AbstractA key step in the development of new pharmaceutical drugs is the identification of the molecular target and distinguishing this from all other gene products that respond indirectly to the drug. Target identification remains a crucial process and a current bottleneck for advancing hits through the discovery pipeline. Here we report a method, that takes advantage of the specific detection of protein–ligand complexes by native mass spectrometry (MS) to probe the protein partner of a ligand in an untargeted method. The key advantage is that it uses unmodified small molecules for binding and, thereby, it does not require labelled ligands and is not limited by the chemistry required to tag the molecule. We demonstrate the use of native MS to identify known ligand–protein interactions in a protein mixture under various experimental conditions. A protein–ligand complex was successfully detected between parthenolide and thioredoxin (PfTrx) in a five-protein mixture, as well as when parthenolide was mixed in a bacterial cell lysate spiked with PfTrx. We provide preliminary data that native MS could be used to identify binding targets for any small molecule.

scholarly journals Chemical proteomics reveals target selectivity of clinical Jak inhibitors in human primary cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
H. Christian Eberl ◽  
Thilo Werner ◽  
Friedrich B. Reinhard ◽  
Stephanie Lehmann ◽  
Douglas Thomson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Dissociation Constants ◽  
Peripheral Blood Mononuclear ◽  
Mass Spectrometry Experiment ◽  
Chemical Proteomics ◽  
Human Primary Cells ◽  
Lipid Kinases ◽  
Single Mass

Abstract Kinobeads are a set of promiscuous kinase inhibitors immobilized on sepharose beads for the comprehensive enrichment of endogenously expressed protein kinases from cell lines and tissues. These beads enable chemoproteomics profiling of kinase inhibitors of interest in dose-dependent competition studies in combination with quantitative mass spectrometry. We present improved bead matrices that capture more than 350 protein kinases and 15 lipid kinases from human cell lysates, respectively. A multiplexing strategy is suggested that enables determination of apparent dissociation constants in a single mass spectrometry experiment. Miniaturization of the procedure enabled determining the target selectivity of the clinical BCR-ABL inhibitor dasatinib in peripheral blood mononuclear cell (PBMC) lysates from individual donors. Profiling of a set of Jak kinase inhibitors revealed kinase off-targets from nearly all kinase families underpinning the need to profile kinase inhibitors against the kinome. Potently bound off-targets of clinical inhibitors suggest polypharmacology, e.g. through MRCK alpha and beta, which bind to decernotinib with nanomolar affinity.

Mapping The HLA Ligandome Of Chronic Lymphocytic Leukemia – Towards Peptide Based Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4123-4123 ◽  
Author(s):  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Claudia Berlin ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Cd8 T Cells ◽  
Target Identification ◽  
Peptide Vaccine ◽  
Hla Class I ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Class I ◽  
Hla Ligands

Abstract Accelerated clonal evolution and inhibition of immune effector functions are fundamental drawbacks of chemotherapeutic treatment of chronic lymphocytic leukemia (CLL) which contribute to increased clinical aggressiveness of relapsed disease. Anticancer immune responses such as graft versus leukemia effects and remissions after donor lymphocyte infusions, on the other hand, have been correlated to long-term CLL-free survival. Clonotype analysis in these cases suggested clonally expanded CD8+ T cells recognizing tumor associated antigens (TAAs) presented by HLA as mediators of the observed effects, thus making CLL an attractive target for peptide vaccine-based immunotherapy. We here report on our approach of direct isolation and identification of naturally processed and presented HLA ligands from tissues of interest by affinity chromatography and mass spectrometry. Comparative and semi-quantitative analysis of the HLA ligandomes of malignant and benign samples provided the rationale for the identification of ligandome derived TAAs (LiTAAs) and informed selection of peptide vaccine candidates. HLA class I ligands were isolated from MACS-sorted CLL cells as well as from normal B cells or PBMC of healthy volunteers using a standard immunoaffinity purification protocol. Liquid chromatography coupled mass spectrometry (LC-MS/MS) peptide analysis was performed on a LTQ Orbitrap hybrid mass spectrometer followed by database assisted processing of fragment spectra. Semi-quantitative data analysis provided information regarding the abundance of HLA ligands in the respective ligandomes. In addition, HLA surface expression on CLL cells and autologous normal B cells was quantitatively determined using a flow cytometric assay. Selected peptides were characterized functionally in IFN-γ ELISPOT assays using PBMC of healthy volunteers and CLL patients. No significant difference in HLA class I surface expression between CLL cells and autologous normal B cells was observed. So far, we were able to map the HLA class I peptidomes of 25 CLL patients and 35 healthy controls. In total, we were able to identify more than 25,000 different HLA ligands representing >8,500 different source proteins. More than 15,000 different ligands were derived from CLL cells representing a total of 6,500 source proteins. A twofold data mining approach was used to identify both, broadly presented LiTAAs suited for off-the-shelf vaccine development, and LiTAAs showing patterns of patient-specific overrepresentation allowing for actively personalized target identification. The former strategy enabled us to pinpoint the most frequently and abundantly represented targets from the bulk of over 2,000 source proteins, which were exclusively represented in the ligandomes of CLL cells. Several published CLL-associated antigens/epitopes were found to be presented (e.g. Pim-1 Oncogene, SET nuclear oncogene, Mucin-1), which served to validate our methodological approach as proof of principle. Beyond that we identified a vast array of novel proteins that are broadly and exclusively represented in the HLA peptidome of CLL cells. Based on these findings we selected HLA-A*02, A*03 and B*07 restricted ligands derived from top ranking LiTAAs (e.g.TP53I11, PARP3, CDCA7L) for immunological characterization. Using patient PBMC, we observed frequent, reproducible and specific immune recognition of the selected peptides by CD8+ T cells in recall ELISPOT assays. The observed reactivity to CLL-associated self-peptides indicates their potential as therapeutic vaccines while underlining the validity of our target identification and selection strategy. Currently we are expanding our analyses to cover a comprehensive spectrum of HLA types with the goal to develop a clinically applicable CLL-specific multi-peptide vaccine. Disclosures: No relevant conflicts of interest to declare.

ChemInform Abstract: Application of Mass Spectrometry for Target Identification and Characterization

ChemInform ◽  
2010 ◽  
Vol 30 (41) ◽  
pp. no-no
Author(s):  
Joseph A. Loo ◽  
Dana E. DeJohn ◽  
Ping Du ◽  
Tracy I. Stevenson ◽  
Rachel R. Ogorzalek Loo
Keyword(s):  
Mass Spectrometry ◽  
Target Identification ◽  
Identification And Characterization

Target identification of natural and traditional medicines with quantitative chemical proteomics approaches

2016 ◽  
Vol 162 ◽  
pp. 10-22 ◽  
Author(s):  
Jigang Wang ◽  
Liqian Gao ◽  
Yew Mun Lee ◽  
Karunakaran A. Kalesh ◽  
Yong Siang Ong ◽  
...  
Keyword(s):  
Target Identification ◽  
Chemical Proteomics ◽  
Traditional Medicines ◽  
Quantitative Chemical

Application of mass spectrometry for target identification and characterization

1999 ◽  
Vol 19 (4) ◽  
pp. 307-319 ◽  
Author(s):  
Joseph A. Loo ◽  
Dana E. DeJohn ◽  
Ping Du ◽  
Tracy I. Stevenson ◽  
Rachel R. Ogorzalek Loo
Keyword(s):  
Mass Spectrometry ◽  
Target Identification ◽  
Identification And Characterization

Drug Target Identification Using Affinity Core-Shell Magnetic Nanoparticles and Mass Spectrometry

2013 ◽  
Vol 31 (6) ◽  
pp. 715-720 ◽  
Author(s):  
Yunhuan Wei ◽  
Tongdan Wang ◽  
Chao Liu ◽  
Qianqian Zhang ◽  
Lishun Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Magnetic Nanoparticles ◽  
Drug Target ◽  
Target Identification ◽  
Core Shell ◽  
Drug Target Identification
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