High-throughput Mass Spectrometry Screening Research Articles

Using a peptide-based mass spectrometry approach to quantitate proteolysis of an intact heterogeneous procollagen substrate by BMP1 for antagonistic antibody screening.

Proteases are critical proteins involved in cleaving substrates that may impact biological pathways, cellular processes, or disease progression. In the biopharmaceutical industry, modulating the levels of protease activity is an important strategy for mitigating many types of diseases. While a variety of analytical tools exist for characterizing substrate cleavages, in vitro functional screening for antibody inhibitors of protease activity using physiologically relevant intact protein substrates remains challenging. In addition, detecting such large protein substrates with high heterogeneity using high-throughput mass spectrometry screening has rarely been reported in the literature with concerns for assay robustness and sensitivity. In this study, we established a peptide-based in vitro functional screening assay for antibody inhibitors of mouse bone morphogenic protein 1 (mBMP1) metalloprotease using a heterogeneous recombinant 66-kDa mouse Procollagen I alpha 1 chain (mProcollagen) substrate. We compared several analytical tools including capillary gel electrophoresis Western blot (CE-Western blot), as well as both intact protein and peptide-based mass spectrometry (MS) to quantitate the mBMP1 proteolytic activity and its inhibition by antibodies using this heterogeneous mProcollagen substrate. We concluded that the peptide-based mass spectrometry screening assay was the most suitable approach in terms of throughput, sensitivity, and assay robustness. We then optimized our mBMP1 proteolysis reaction after characterizing the enzyme kinetics using the peptide-based MS assay. This assay resulted in Z' values ranging from 0.6 to 0.8 from the screening campaign. Among over 1200 antibodies screened, IC50 characterization was performed on the top candidate hits, which showed partial or complete inhibitory activities against mBMP1.

Identification of the first highly selective inhibitor of human lactate dehydrogenase B

Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

A direct peptide reactivity assay using a high-throughput mass spectrometry screening platform for detection of skin sensitizers

Chemical-peptide conjugation is the molecular initiating event in skin sensitization. The OECD test guideline uses a high-performance liquid chromatography/ultraviolet (HPLC/UV) detection method to quantify chemical-peptide conjugation in a direct peptide reactivity assay (DPRA), which measures the depletion of two synthetic peptides containing lysine or cysteine residues. To improve assay throughput, sensitivity and accuracy, an automated 384-well plate-based RapidFire solid-phase extraction (SPE) system coupled with tandem mass spectrometry (MS/MS) DPRA was developed and validated in the presence of a newly designed internal standard. Compared to the HPLC/UV-based DPRA, the automated SPE-MS/MS-based DPRA improved throughput from 16 min to 10 s per sample, and substrate peptides usage was reduced from 100 mM to 5 μM. When implementing the SPE-MS/MS-based DPRA into a high-throughput platform, we found 10 compounds that depleted lysine peptide and 24 compounds that depleted cysteine peptide (including 7 unreported chemicals from 55 compounds we tested) in a concentration-response manner. The adduct formation between cysteine and cinnamic aldehyde and ethylene glycol dimethacrylate were further analyzed using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) to confirm the conjugation. Overall, the automated SPE-MS/MS-based platform is an efficient, economic, and accurate way to detect skin sensitizers.

A high throughput mass spectrometry screening analysis based on two-dimensional carbon microfiber fractionation system

A novel high-throughput, solvent saving and versatile integrated two-dimensional microscale carbon fiber/active carbon fiber system (2DμCFs) that allows a simply and rapid separation of compounds in low-polar, medium-polar and high-polar fractions, has been coupled with ambient ionization-mass spectrometry (ESI-Q-TOF-MS and ESI-QqQ-MS) for screening and quantitative analyses of real samples. 2DμCFs led to a substantial interference reduction and minimization of ionization suppression effects, thus increasing the sensitivity and the screening capabilities of the subsequent MS analysis. The method has been applied to the analysis of Schisandra Chinensis extracts, obtaining with a single injection a simultaneous determination of 33 compounds presenting different polarities, such as organic acids, lignans, and flavonoids in less than 7min, at low pressures and using small solvent amounts. The method was also validated using 10 model compounds, giving limit of detections (LODs) ranging from 0.3 to 30ngmL−1, satisfactory recoveries (from 75.8 to 93.2%) and reproducibilities (relative standard deviations, RSDs, from 1.40 to 8.06%).

Application of MALDI‐MS analysis of Rainforest chemodiversity: a keystone for biodiversity conservation and sustainable use

Brazil hosts the largest proportion of global biodiversity and has demonstrated its commitment in conservation and sustainable use being a key negotiator of the Nagoya Protocol. The “Convention on Biological Diversity” (CBD) calls for actions to reduce extinction rates, something that according with different theories is of fundamental importance for the survival of life on Earth. Contrary to its position in the CBD meetings, Brazil approved a new Forest Code that will result in escalating deforestation, increasing the urgency to demonstrate the value of native species. Extractive-based activities of forest inhabitants are basically economical prospective activities upon the forest richness whose act harmfully against the environment. For centuries, such activity resulted in low profits, triggering a perverse logic that profit increase is necessarily linked to extraction increase. In the last decades, new strategies, as for instance the Sustainable Development Reserve Mamiraua, are showing that forest preservation can also be profitable. Considering the new paradigm of green economy, which now surrounds all this tensioned discussion, we are bringing to the eyesight of policy-makers results on biodiversity conservation research: combining floristic and chemodiversity surveys, using fast high throughput mass spectrometry screening (HT-MSS), to screen forest leaves for economically valued natural products. Matrix-assisted laser desorption/ionization (MALDI) ionizationbased machines are well known by their ability to furnish fast data and can be an important tool for HT-MSS. One of the aims of a long-term ongoing BIOTA/FAPESP research project at the Serra do Mar State Park is to understand ecophysiological traits of leaves, and MALDI-MS was an alternative to identify alkaloids as one of the nitrogen sink. Regarding the following main points: (1) analysis of plant attributed nitrogen fixation and (2) profitable policies for biodiversity conservation, we suggest the screening for alkaloids to be carried out using the leaves of the trees that are top ranked in population density lists within the forest sociology. In order to reach these aims, a set larger than 500 samples was screened for the occurrence of alkaloids and botanical identification. Classical chemical procedures were also applied to validate the results (Dragendorff, Mayer and Wagner assays). In this letter, we provide the first HT MALDI-MS and MALDI-MS/MS forest screening method to provide added value to local specimens of plants. Ionic liquid was used to get around known ionization problems of small molecules (< 1200Da) by avoiding isobaric ions formation by the matrix. Although, LC-ESI-MS is the common technique to screen small molecules, it is highly time consuming, since in six days, only 50 samples can be analyzed. Therefore, our strategy is based on the ability of MALDI to quickly screen a very large number of samples using an ionic liquid as matrix. Using this approach, we can screen up to 200 samples within a 3 h time-frame.

High content secondary screening assay to model NRTI-induced mitochondrial toxicity

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (Km = 10.5 μM). The assay was highly reproducible with an average Z′ value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC50 values of 0.88 and 0.12 μM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 μM – 14% conformation rate). Of the confirmed hits 172 had IC50 values of <10 μM, including 111 <1 μM and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.

Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate ( K m = 10.5 μM). The assay was highly reproducible with an average Z′ value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC 50 values of 0.88 and 0.12 μM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 μM – 14% conformation rate). Of the confirmed hits 172 had IC 50 values of <10 μM, including 111 <1 μM and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further medicinal chemistry and characterization of SCD inhibitors should lead to the development of reagents to treat metabolic disorders.

High-Throughput Mass Spectrometry Screening for Inhibitors of Phosphatidylserine Decarboxylase

A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries. The high-throughput mass spectrometry assay directly measures phosphatidylserine and phosphatidylethanolamine using the RapidFiretrade mark platform at a rate of 1 sample every 7.5 s. The assay is robust, with an average Z' value of 0.79 from a screen of 9920 compounds. Of 60 compounds selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry permitted a high-quality screen to be performed for an otherwise intractable target.