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Direct screening of enzyme activity using infrared matrix‐assisted laser desorption electrospray ionization
Author(s) -
Nazari Milad,
Ekelöf Måns,
Khodjaniyazova Sitora,
Elsen Nathaniel L.,
Williams Jon D.,
Muddiman David C.
Publication year - 2017
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7971
Subject(s) - desorption electrospray ionization , mass spectrometry , chemistry , desorption , electrospray ionization , matrix (chemical analysis) , chromatography , matrix assisted laser desorption/ionization , analytical chemistry (journal) , sample preparation , ionization , organic chemistry , chemical ionization , adsorption , ion
Rationale High‐throughput screening (HTS) is a critical step in the drug discovery process. However, most mass spectrometry (MS)‐based HTS methods require sample cleanup steps prior to analysis. In this work we present the utility of infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) for monitoring an enzymatic reaction directly from a biological buffer system with no sample cleanup and at high throughput. Methods IR‐MALDESI was used to directly analyze reaction mixtures from a well plate at different time points after reaction initiation. The percent conversion of precursors to products was used to screen the enzyme activity. The reaction was performed with two different concentrations of precursors and enzyme in order to assess the dynamic range of the assay. Eventually, a pseudo‐HTS study was designed to investigate the utility of IR‐MALDESI screening enzyme activity in a high‐throughput manner. Results IR‐MALDESI was able to readily monitor the activity of IDH1 over time at two different concentrations of precursors and enzyme. The calculated Z‐factors of 0.65 and 0.41 confirmed the suitability of the developed method for screening enzyme activity in HTS manner. Finally, in a single‐blind pseudo‐HTS analysis IR‐MALDESI was able to correctly predict the identity of all samples, where 8/10 samples were identified with high confidence and the other two samples with lower confidence. Conclusions The enzymatic activity of IDH1 was screened by directly analyzing the reaction content from the buffer in well plates with no sample cleanup steps. This proof‐of‐concept study demonstrates the robustness of IR‐MALDESI for direct analysis of enzymatic reactions from biological buffers with no sample cleanup and its immense potential for HTS applications.