A matrix-assisted laser desorption/ionization tandem mass spectrometry method for direct screening of small molecule mixtures against an aminoglycoside kinase

Smith, Anne Marie; Awuah, Emelia; Capretta, Alfredo; Brennan, John D.

doi:10.1016/j.aca.2013.05.027

Cited by 9 publications

(19 citation statements)

References 40 publications

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“…However, the error bars for the IC 50 plots (n = 3) tend to show a large error for some points, possibly owing to variations in matrix crystallization, which is a common issue for MALDI-MS. The novel inhibitor EME 467, found in previous work by our group to have a K I value of (1.2 AE 0.6) mm, [24] was found in the current assay to have a K I value of (1.1 AE 0.2) mm ( …”

Section: Quantitative Dose-dependent Response Curvesmentioning

confidence: 48%

“…Dual-kinase assay conditions: Reaction conditions were similar to our previous report, [24] with minor changes. Briefly, all assays were performed by pre-incubating the enzymes (APH3'IIIa and/or PKA) with cofactors (Mg(OAc) 2 and ATP) for 5 min at 37 8C.…”

Section: Methodsmentioning

confidence: 99%

“…[24] Operation of the AP-MALDI ion trap system in internal timing mode with the use of a set internal integration time (24 s) and delay time (90 s) reduced background interferences from matrix ions and allowed for the production of peaks that could be integrated in a manner similar to that of HPLC injection peaks. This approach significantly reduced variability in the signal and also improved overall signal levels.…”

Section: Validation Of Assaymentioning

confidence: 99%

“…Assay throughput was increased by screening both enzyme reactions simultaneously against mixtures of potential inhibitors rather than discrete compounds. [24,26,27] A total of 21 mixtures were screened in duplicate with 10 compounds per mixture, including known inhibitors of both enzyme systems as well as +ve (enzyme + substrate) and Àve (no enzyme) controls. Although the mixture could have been made more complex so that it contained 20 or more compounds instead of 10, due to the use of a kinase-directed library and a potentially high hit rate, a value of 10 compounds per mixture was chosen to avoid having a hit in each mixture.…”

Section: Simultaneous Screening Of Mixturesmentioning

confidence: 99%

“…MS, absorbance, fluorescence, etc. ), [21,[23][24][25] possibly due to compounds increasing enzyme activity or en- hancing signals as a result of interference (e.g. enhanced ionization).…”

Section: Simultaneous Screening Of Mixturesmentioning

confidence: 99%

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Simultaneous Inhibition Assay for Human and Microbial Kinases via MALDI‐MS/MS

Smith

Brennan

2014

ChemBioChem

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Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution.

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Section: Quantitative Dose-dependent Response Curvesmentioning

confidence: 48%

Section: Methodsmentioning

confidence: 99%

Section: Validation Of Assaymentioning

confidence: 99%

Section: Simultaneous Screening Of Mixturesmentioning

confidence: 99%

“…MS, absorbance, fluorescence, etc. ), [21,[23][24][25] possibly due to compounds increasing enzyme activity or en- hancing signals as a result of interference (e.g. enhanced ionization).…”

Section: Simultaneous Screening Of Mixturesmentioning

confidence: 99%

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Simultaneous Inhibition Assay for Human and Microbial Kinases via MALDI‐MS/MS

Smith

Brennan

2014

ChemBioChem

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High‐Throughput Mass Spectrometry Complements Protein Engineering

Xue

Choe

et al. 2021

Protein Engineering

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

Harvey

2018

Mass Spectrometry Reviews

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This review is the eighth update of the original article published in 1999 on the application of Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2014. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly- saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2018 Wiley Periodicals, Inc. Mass Spec Rev 37:353-491, 2018.

show abstract

A matrix-assisted laser desorption/ionization tandem mass spectrometry method for direct screening of small molecule mixtures against an aminoglycoside kinase

Cited by 9 publications

References 40 publications

Simultaneous Inhibition Assay for Human and Microbial Kinases via MALDI‐MS/MS

Simultaneous Inhibition Assay for Human and Microbial Kinases via MALDI‐MS/MS

High‐Throughput Mass Spectrometry Complements Protein Engineering

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

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