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

Abstract

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.

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

“…Subsequently, ADP is further degraded to AMP and finally to adenosine (Figure ). Thus, mass spectrometric detection has the potential to identify enzymatically generated intermediates that would likely be disregarded with use of conventional spectroscopic methods …”

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

“…In setup 1 (Figure 1, middle trace: light grey dashed line), the enzymatic activity of iAP was measured towards its nucleotide substrate AMP. Activity changes in AMP substrate degradation along with adenosine (Ado) product generation, caused by the introduction of additional AMP substrate as well as increasing concentrations of the competitive nucleotide substrates ADP and ATP [15], were investigated. Concentration of the enzyme and the AMP substrate introduced to the system were 2.4 U/mL and 80 µM, respectively.…”

“…Enzyme and substrate concentrations were chosen to obtain an enzymatic substrate conversion rate that results in a sufficient mass spectrometric substrate as well as product trace intensity. The injection of different concentrations of substrate AMP or alternative substrates ADP or ATP led to the generation of intermediates and an altered Ado product generation, which is due to the enzyme´s ability to dephosphorylate its substrates in a stepwise manner [15].…”

Mass spectrometric real‐time monitoring of an enzymatic phosphorylation assay using internal standards and data‐handling freeware

“…However, several authors have described continuous-flow [24,25] approaches for enzyme assays, like on-line digestion by immobilized trypsin [32][33][34] or other immobilized enzyme reactors, like protein tyrosine phosphatase, [35] glucose oxidase, [36] and alkaline phosphatase. [37][38][39] As utilized in this study, similar on-line reaction detection systems were used in earlier publications to monitor acetylcholinesterase inhibition, [40][41][42] enzymatic hydrolysis, [43][44][45] solvolysis, [45] disulfide reduction [45] and protein unfolding. [46][47][48] However, the data obtained could not be calculated to quantitative values, due to the lack of using internal standards.…”