Targeted Mass Spectrometry-Based Approach for Protein–Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy

Jin, Lorrain; Wang, Dongyu; Gooden, David M.; Ball, Carol H.; Fitzgerald, Michael C.

doi:10.1021/acs.analchem.6b02658

Cited by 12 publications

(12 citation statements)

References 22 publications

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“…Generated peptides are then analysed through mass spectrometry to evaluate selective methionine oxidation. Analyses of oxidised and non-oxidised methionine-containing peptides versus the guanidinium hydrochloride concentration reveal that proteins bound to ligands show a larger transition midpoint shift than control samples [ 13 , 135 , 136 , 137 ]. Indeed, several target proteins of compounds such as resveratrol and cyclophilin A were verified using SPROX [ 133 , 137 , 138 ].…”

Section: Natural Products Drug Discovery Research and Development mentioning

confidence: 99%

Natural Products for Drug Discovery in the 21st Century: Innovations for Novel Drug Discovery

Thomford

Senthebane

Rowe

et al. 2018

IJMS

796

514

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The therapeutic properties of plants have been recognised since time immemorial. Many pathological conditions have been treated using plant-derived medicines. These medicines are used as concoctions or concentrated plant extracts without isolation of active compounds. Modern medicine however, requires the isolation and purification of one or two active compounds. There are however a lot of global health challenges with diseases such as cancer, degenerative diseases, HIV/AIDS and diabetes, of which modern medicine is struggling to provide cures. Many times the isolation of “active compound” has made the compound ineffective. Drug discovery is a multidimensional problem requiring several parameters of both natural and synthetic compounds such as safety, pharmacokinetics and efficacy to be evaluated during drug candidate selection. The advent of latest technologies that enhance drug design hypotheses such as Artificial Intelligence, the use of ‘organ-on chip’ and microfluidics technologies, means that automation has become part of drug discovery. This has resulted in increased speed in drug discovery and evaluation of the safety, pharmacokinetics and efficacy of candidate compounds whilst allowing novel ways of drug design and synthesis based on natural compounds. Recent advances in analytical and computational techniques have opened new avenues to process complex natural products and to use their structures to derive new and innovative drugs. Indeed, we are in the era of computational molecular design, as applied to natural products. Predictive computational softwares have contributed to the discovery of molecular targets of natural products and their derivatives. In future the use of quantum computing, computational softwares and databases in modelling molecular interactions and predicting features and parameters needed for drug development, such as pharmacokinetic and pharmacodynamics, will result in few false positive leads in drug development. This review discusses plant-based natural product drug discovery and how innovative technologies play a role in next-generation drug discovery.

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Section: Natural Products Drug Discovery Research and Development mentioning

confidence: 99%

Natural Products for Drug Discovery in the 21st Century: Innovations for Novel Drug Discovery

Thomford

Senthebane

Rowe

et al. 2018

IJMS

796

514

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“…However, the protein hits identified using such a thermodynamic profiling strategy do need further validation in clinical samples before their translation into diagnostic or therapeutic practice. A promising validation strategy is the recently developed targeted mass spectrometry-based SPROX approach for the detection and quantitation of target methionine-containing peptides, 46 which will help validate the changes of protein folding stability observed in the cell culture models of the disease.…”

Section: Discussionmentioning

confidence: 99%

Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins Using SILAC-SPROX

Liu

Fitzgerald

2017

J. Proteome Res.

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Proteomic methods for disease state characterization and biomarker discovery have traditionally utilized quantitative mass spectrometry methods to identify proteins with altered expression levels in disease states. Here we report on the large-scale use of protein folding stability measurements to characterize different subtypes of breast cancer using the Stable Isotope Labeling with Amino Acids in Cell Culture and Stability of Proteins from Rates of Oxidation (SILAC-SPROX) technique. Protein folding stability differences were studied in a comparison of two luminal breast cancer subtypes, luminal-A and -B (i.e., MCF-7 and BT-474 cells, respectively), and in a comparison of a luminal-A and basal subtype of the disease (i.e., MCF-7 and MDA-MB-468 cells, respectively). The 242 and 445 protein hits identified with altered stabilities in these comparative analyses, included a large fraction with no significant expression level changes. This suggests thermodynamic stability measurements create a new avenue for protein biomarker discovery. A number of the identified protein hits are known from other biochemical studies to play a role in tumorigenesis and cancer progression. This not only substantiates the biological significance of the protein hits identified using the SILAC-SPROX approach, but it also helps elucidate the molecular basis for their disregulation and/or disfunction in cancer.

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“…A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 . Briefly, 70 uL aliquots of a 1 mg/mL solution of recombinant YBX1 in 20mM phosphate buffer (pH 7.4) were combined with either 7.7 μL of a 3 mM TAM solution prepared in DMSO or 7.7 μL of DMSO to generate the (+) and (−) ligand samples, respectively.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

Ogburn

Jin

et al. 2017

J. Proteome Res.

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The proteins in an MCF-7 cell line were probed for tamoxifen (TAM) and n-desmethyl tamoxifen (NDT) induced stability changes using the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with two different quantitative proteomics strategies, including one based on SILAC and one based on isobaric mass tags. Over 1000 proteins were assayed for TAM- and NDT- induced protein stability changes, and a total of 163 and 200 protein hits were identified in the TAM and NDT studies, respectively. A subset of 27 high confidence protein hits were reproducibly identified with both proteomics strategies and/or with multiple peptide probes. One-third of the high confidence hits have previously established experimental links to the estrogen receptor, and nearly all of the high confidence hits have established links to breast cancer. One high confidence protein hit that has known estrogen receptor binding properties, Y-box binding protein 1 (YBX1), was further validated as a direct binding target of TAM using both the SPROX and pulse proteolysis techniques. Proteins with TAM- and/or NDT-induced expression level changes were also identified in the SILAC-SPROX experiments. These proteins with expression level changes included only a small fraction of those with TAM- and/or NDT-induced stability changes.

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Targeted Mass Spectrometry-Based Approach for Protein–Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy

Cited by 12 publications

References 22 publications

Natural Products for Drug Discovery in the 21st Century: Innovations for Novel Drug Discovery

Natural Products for Drug Discovery in the 21st Century: Innovations for Novel Drug Discovery

Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins Using SILAC-SPROX

Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

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