Real-time ESI-MS of Enzymatic Conversion: Impact of Organic Solvents and Multiplexing

Scheerle, Romy K.; Graßmann, Johanna; Letzel, Thomas

doi:10.2116/analsci.28.607

Cited by 11 publications

(11 citation statements)

References 42 publications

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“…The most suitable combination was identified as that which resulted in a complete degradation of the substrate within 30 min and therewith in a considerable flattening of ATP substrate trace. iAP assays were performed in positive ionization mode, to maintain comparability to other enzymatic assays already established and in this context to prospectively conduct experiments with multiplex enzymatic assays . Even though higher intensities may be anticipated in negative mode, initial intensities of the nucleotides substrates between at least 150 000 counts for ATP substrate in pH 9.0 and up to 400 000 counts for AMP in pH 7.4 were obtained in positive mode.…”

Section: Methodsmentioning

confidence: 99%

Utilization of real‐time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase

Kaufmann

Graßmann

Treutter

et al. 2014

Rapid Comm Mass Spectrometry

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The comparison of colorimetric and mass spectrometric methods to elucidate enzyme kinetics and specificity clearly underlines the advantages of mass spectrometric detection for the investigation of complex multi-component enzymatic assays. The entire course of enzymatic substrate degradation was revealed with different nucleotide substrates, thus allowing a specific monitoring of intestinal alkaline phosphatase activity.

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Section: Methodsmentioning

confidence: 99%

Utilization of real‐time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase

Kaufmann

Graßmann

Treutter

et al. 2014

Rapid Comm Mass Spectrometry

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“…Moreover enzymatic activities were found to be almost as high in the multiplex approach as in the single assays (78 % for chitinase and 94 % for chymotrypsin). Further examples can be found in Scheerle et al …”

Section: Determination Of Reaction Profiles and Cleavage Specificitiementioning

confidence: 94%

“…Beyond this, most chromatographic columns require the addition of at least small proportions of organic solvents to maintain stability. On the other hand, organic solvents affect enzymatic activity, through denaturation, interference with substrate binding, direct inhibition, and other negative effects . For this reason various solvents have to be tested for their influence on the enzyme(s) of interest before application in the mixing assay .…”

Section: Investigation Of Inhibitors By Using a Continuous‐flow Assaymentioning

confidence: 99%

“…On the other hand, organic solvents affect enzymatic activity, through denaturation, interference with substrate binding, direct inhibition, and other negative effects . For this reason various solvents have to be tested for their influence on the enzyme(s) of interest before application in the mixing assay . In order to maintain constant organic solvent exposure to the enzyme, isocratic separation over the entire measurement time is favored, to ensure a consistent substrate and product signal .…”

Section: Investigation Of Inhibitors By Using a Continuous‐flow Assaymentioning

confidence: 99%

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Enzymatic Assays Coupled with Mass Spectrometry with or without Embedded Liquid Chromatography

et al. 2015

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This article reviews monitoring strategies for enzymatic assays coupled with mass spectrometric detection. This coupling has already been shown to be helpful in providing versatile and detailed knowledge about enzyme kinetics. Various available publications address two general approaches. 1) The continuous-flow setup allows real-time determination of substrate degradation. Simultaneously, resulting product or potential intermediates can be detected. 2) The online coupled continuous-flow mixing assay allows the direct coupling of an enzymatic assay to chromatographic separation of complex mixtures. The latest efforts in improving the methodology have been made with regard to miniaturization. This is especially advantageous with regard to reducing costly consumption of chemicals. Finally, these developments are applicable for diverse bioanalytical purposes in the realms of pharmaceutical, biotechnological, food, and environmental research.

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“…In setup 2 an isocratic eluent flow with 90% 10 mM ammonium acetate pH 7.4 with 10% methanol was used to separate the sample on a high temperature-compatible Zirchrom PBD column (100x1 mm, 3 µm) (ZirChrom Separations, Inc., Anoka, MN, USA), which was integrated into a column oven (HT HPLC 2000, Scientific Instruments Manufacturer GmbH, Oberhausen, Germany) ( Figure 1, middle trace). The organic solvent concentration tolerable for AChE was preliminarily tested [17], whereupon 10% methanol was found to be suitable to maintain sufficient enzymatic activity. A temperature gradient was applied to improve the chromatographic separation.…”

Section: Instrumentationmentioning

confidence: 99%

Achroma Software-High-Quality Policy in (a-)Typical Mass Spectrometric Data Handling and Applied Functional Proteomics

Krappmann¹,

Kaufmann²,

Scheerle³

et al. 2014

J Proteomics Bioinform

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Data evaluation of mass spectrometric raw data is an essential step to obtain high-quality results. In our days an exorbitant amount of raw data are produced in analytical (bio)chemistry due to the utilization of sophisticated experimental setups. The recently published free software Achroma has been developed to overcome increasing data processing challenges by providing the possibility of a comprehensive data analysis (http://openmasp.hswt. de/pages/project/achroma.php). To illustrate (a-)typical data evaluation, an online coupled continuous flow system hyphenated with mass spectrometric detection was applied to investigate enzymatic activity changes in the presence of regulatory molecules and alternative substrates. The extended software strategy, the processing as well as evaluation of data is presented in detail based on enzymatic assays of intestinal alkaline phosphatase (iAP) and acetylcholine esterase (AChE). Different Achroma data evaluation modules enabled a high quality analysis. This included the elucidation of enzymatic substrate preferences by means of the calculation of negative and positive peak areas as well as the identification of an inhibitory molecule by comparing different mass spectra with regard to their overall composition. The possibility of an automatically performed validity control to monitor the systems robustness furthermore emphasized the usefulness of Achroma software regarding its applicability in the area of 'functional proteomics' data handling.

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Real-time ESI-MS of Enzymatic Conversion: Impact of Organic Solvents and Multiplexing

Cited by 11 publications

References 42 publications

Utilization of real‐time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase

Utilization of real‐time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase

Enzymatic Assays Coupled with Mass Spectrometry with or without Embedded Liquid Chromatography

Achroma Software-High-Quality Policy in (a-)Typical Mass Spectrometric Data Handling and Applied Functional Proteomics

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