TLC-UV hyphenated with MALDI-TOFMS for the screening of invertase substrates in plant extracts

Abstract

In this study, thin-layer chromatography (TLC) hyphenated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was developed for the screening of invertase substrates in complex matrices. BfrA, a specific β-D-fructofuranosidase from Leishmania major, was chosen as a model enzyme to screen biological activity in plant extracts due to its capacity to hydrolyze specific carbohydrates. TLC was considered to be a reliable technique for screening substrates (bioactive molecules) in plant extracts due to its quantitative capabilities whereas MALDI-TOFMS was particularly useful for rapid identification. The first part of this approach consisted of a differential analysis by TLC-densitometry to highlight band under- and over-expressions in plant extract between blank and enzymatic reaction. Zones of interest were then immediately analyzed by TLC-MALDI-TOFMS coupling to identify bioactive molecules. Development of the method presented various challenges: the separation and analysis of isomers (such as glucose and fructose), the high matrix effect (demonstrated by the analysis of products with invertase enzyme naturally present in plant extract), and the analysis of polar molecules with low molecular mass (sugars). Thanks to the separative technique, the specificity of detection, and the high precision of the characterization, this method was shown to be feasible for the analysis of bioactive molecules in complex mixtures containing interfering compounds (e.g. proteins, salts). Overall, this study demonstrates that Thin-layer chromatography hyphenated with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a simple, rapid, precise and efficient method for the analysis of suitable substrates in raw samples.

Keywords: Invertase; Matrix assisted laser desorption-ionization time of flight mass spectrometry; Plant extracts; Thin-layer chromatography.