Biochemical Assays of Immobilized Oligonucleotides with Mass Spectrometry

Langmuir ◽  
2008 ◽  
Vol 24 (10) ◽  
pp. 5433-5438 ◽  
Author(s):  
Haim Tsubery ◽  
Milan Mrksich
Keyword(s):  
Mass Spectrometry ◽  
Biochemical Assays ◽  
Immobilized Oligonucleotides

Mass spectrometry-based biochemical assays for enzyme-inhibitor screening

2007 ◽  
Vol 26 (9) ◽  
pp. 867-883 ◽  
Author(s):  
Arjen R. de Boer ◽  
Henk Lingeman ◽  
Wilfried M.A. Niessen ◽  
Hubertus Irth
Keyword(s):  
Mass Spectrometry ◽  
Enzyme Inhibitor ◽  
Inhibitor Screening ◽  
Biochemical Assays

scholarly journals Comparison of Liquid Chromatography/Mass Spectrometry, ELISA, and Phosphatase Assay for the Determination of Microcystins in Blue-Green Algae Products

2001 ◽  
Vol 84 (4) ◽  
pp. 1035-1044 ◽  
Author(s):  
James F Lawrence ◽  
Barbara Niedzwiadek ◽  
Cathie Menard ◽  
Benjamin P Y Lau ◽  
David Lewis ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Green Algae ◽  
Solid Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blue Green Algae ◽  
Biochemical Assays ◽  
Liquid Chromatography Tandem Mass ◽  
Protein Phosphatase Inhibition ◽  
Phosphatase Assay

Abstract More than 100 samples of blue-green algae products (consisting of Aphanizomenon, Spirulina, and unidentified blue-green algae) in the form of pills, capsules, and powders were collected from retail outlets from across Canada. The samples were extracted with 75% methanol in water and centrifuged to remove solids. Aliquots of the extracts along with spiked blank sample extracts were sent to each participating laboratory and independently analyzed for microcystins by enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay, and by liquid chromatography–tandem mass spectrometry (LC–MS/MS) after sample cleanup using C18 solid–phase extraction. The results obtained by ELISA and LC–MS/MS agreed very well over a concentration range of about 0.5–35 μg/g. The colorimetric phosphatase results generally agreed with the other 2 methods. While the 2 biochemical assays measured total microcystin content compared with a standard of microcystin LR, the LC–MS/MS method measured specific microcystins (LA, LR, RR, YR) using external standards of these for identification and quantitation. Microcystin LR was found in all positive samples by LC–MS/MS. Microcystin LA was the only other microcystin found in the samples analyzed. These 2 microcystins represent essentially all the microcystins that were present in the extracts. Otherwise, the LC–MS/MS results would have been significantly lower than the results of the biochemical assays had other unknown microcystins been present.

scholarly journals A Robust Multiplex Mass Spectrometric Assay for Screening Small-Molecule Inhibitors of CD73 with Diverse Inhibition Modalities

2018 ◽  
Vol 23 (3) ◽  
pp. 264-273 ◽  
Author(s):  
Jessica McManus ◽  
Timothy He ◽  
Julie-Ann Gavigan ◽  
Ghislaine Marchand ◽  
Stephanie Vougier ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Dynamic Range ◽  
Elevated Concentration ◽  
Antitumor Effects ◽  
Extracellular Adenosine ◽  
Biochemical Assays ◽  
Small Molecule Libraries

CD73/Ecto-5′-nucleotidase is a membrane-tethered ecto-enzyme that works in tandem with CD39 to convert extracellular adenosine triphosphate (ATP) into adenosine. CD73 is highly expressed on various types of cancer cells and on infiltrating suppressive immune cells, leading to an elevated concentration of adenosine in the tumor microenvironment, which elicits a strong immunosuppressive effect. In preclinical studies, targeting CD73 with anti-CD73 antibody results in favorable antitumor effects. Despite initial studies using antibodies, inhibition of CD73 catalytic activity using small-molecule inhibitors may be more effective in lowering extracellular adenosine due to better tumor penetration and distribution. To screen small-molecule libraries, we explored multiple approaches, including colorimetric and fluorescent biochemical assays, and due to some limitations with these assays, we developed a mass spectrometry (MS)-based assay. Only the MS-based assay offers the sensitivity and dynamic range required for screening small-molecule libraries at a substrate concentration close to the Km value of substrate and for evaluating the mode of binding of screening hits. To achieve a throughput suitable for high-throughput screening (HTS), we developed a RapidFire–tandem mass spectrometry (RF-MS/MS)-based multiplex assay. This assay allowed a large diverse compound library to be screened at a speed of 1536 reactions per 40–50 min.

scholarly journals Strategic Design of Catalytic Lysine-Targeting Reversible Covalent BCR-ABL Inhibitors

2021 ◽  
Author(s):  
David Quach ◽  
Guanghui Tang ◽  
Jothi Anantharajan ◽  
Nithya Baburajendran ◽  
Anders Poulsen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Acquired Resistance ◽  
Kinase Domain ◽  
Label Free ◽  
Single Digit ◽  
X Ray Crystallography ◽  
Strategic Design ◽  
Biochemical Assays ◽  
Covalent Inhibitors ◽  
Reversible Covalent

Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved single-digit nanomolar potency against both wild-type and mutant ABL kinases, especially ABL<sup>T315I</sup> bearing the gatekeeper residue mutation. We show that, by using techniques including mass spectrometry, time-dependent biochemical assays and X-ray crystallography, the evolutionarily conserved lysine can be targeted selectively. Furthermore, we show that the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate potential off-targets of our compounds at proteome-wide level in different cancer cell lines.

scholarly journals Strategic Design of Catalytic Lysine-Targeting Reversible Covalent BCR-ABL Inhibitors

2021 ◽  
Author(s):  
David Quach ◽  
Guanghui Tang ◽  
Jothi Anantharajan ◽  
Nithya Baburajendran ◽  
Anders Poulsen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Acquired Resistance ◽  
Kinase Domain ◽  
Label Free ◽  
Single Digit ◽  
X Ray Crystallography ◽  
Strategic Design ◽  
Biochemical Assays ◽  
Covalent Inhibitors ◽  
Reversible Covalent

Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved single-digit nanomolar potency against both wild-type and mutant ABL kinases, especially ABL<sup>T315I</sup> bearing the gatekeeper residue mutation. We show that, by using techniques including mass spectrometry, time-dependent biochemical assays and X-ray crystallography, the evolutionarily conserved lysine can be targeted selectively. Furthermore, we show that the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate potential off-targets of our compounds at proteome-wide level in different cancer cell lines.

Small Proteins; Big Questions

2021 ◽  
Author(s):  
Todd Gray ◽  
Gisela Storz ◽  
Kai Papenfort
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Ribosome Profiling ◽  
Therapeutic Approaches ◽  
Small Proteins ◽  
Biochemical Assays ◽  
Regulatory Functions

In recent years there has been increased appreciation that a whole category of proteins—small proteins of around 50 amino acids or fewer in length—have been missed by annotation as well as by genetic and biochemical assays. With the increased recognition that small proteins are stable within cells and have regulatory functions, there has been intensified study of these proteins. As a result, important questions about small proteins in bacteria and archaea are coming to the fore. Here, we give an overview of these questions, the initial answers, and the approaches needed to address the questions more fully. More detailed discussions of how small proteins can be identified by ribosome profiling and mass spectrometry approaches are provided by two accompanying reviews. We are excited by the prospects of new insights and possibly therapeutic approaches coming from this emerging field.

Thioredoxin reductase, an emerging target for anticancer metallodrugs. Enzyme inhibition by cytotoxic gold(iii) compounds studied with combined mass spectrometry and biochemical assays

MedChemComm ◽  
2011 ◽  
Vol 2 (1) ◽  
pp. 50-54 ◽  
Author(s):  
Chiara Gabbiani ◽  
Guido Mastrobuoni ◽  
Francesca Sorrentino ◽  
Barbara Dani ◽  
Maria Pia Rigobello ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Enzyme Inhibition ◽  
Thioredoxin Reductase ◽  
Biochemical Assays ◽  
Anticancer Metallodrugs
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