Biotechnology Letters 26: 1025–1030, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 1025 Screening of sugar converting enzymes using quantitative MALDI-ToF mass spectrometry Ditte Bungert 1 , Sabine Bastian 2 , Dorothée M. Heckmann-Pohl 2 , Friedrich Giffhorn 2 , Elmar Heinzle 1 & Andreas Tholey 1,∗ 1 Technische Biochemie and 2 Angewandte Mikrobiologie, Universität des Saarlandes, 66123 Saarbrücken, Germany ∗ Author for correspondence (Fax: +49 (681) 302 4572; E-mail: a.tholey@mx.uni-saarland.de) Received 21 April 2004; Accepted 27 April 2004 Key words: ionic liquid matrices, MALDI-ToF MS, pyranose oxidase, quantitation, screening Abstract Quantitative matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) was applied for the screening of ten pyranose oxidase variants. Quantitative MALDI-ToF MS using isotopic labeled internal standards and ionic liquid matrices was performed using aliquots of enzyme reaction mixtures without prepurification steps. The results obtained were in good agreement with HPLC measurements. Analysis time was approx. 3.5 min for a five-fold determination. Thus, quantitative MALDI-ToF MS can be used as a tool for screening of sugar converting enzymes. Introduction Enzyme-catalyzed reactions have an increasing im- pact in almost all fields of biotechnology, biochemistry and pharmaceutical sciences and production processes (Straathof et al. 2002). Techniques like site-directed mutagenesis, phage-display, error-prone PCR and gene shuffling allow the creation of a huge amount of enzyme variants within a short period of time. The permanently expanding amount of new biocata- lysts necessitates simple analytical methods for a fast screening of variants for possible successful candid- ates (Tholey & Heinzle 2002). Beside well known analytical techniques like HPLC, GC/MS and NMR, which are often very time-consuming, optical meth- ods are most commonly used. However, these meth- ods may suffer from some severe restrictions because chromophoric or fluorophoric properties of either sub- strates or products must change during enzymatic con- version. In a number of biotechnologically interesting enzymatic reactions, substrates or products of interest do not contain such chromophoric or fluorophoric groups. This necessitates the use of artificial sub- strates, which can lead to misleading results. Another drawback of optical screening methods may be the disturbance by other ingredients (buffers, cofactors) in the reaction mixtures. Mass spectrometry (MS) has recently become an important for the analysis of both large and small bio- molecules such as proteins, peptides, amino acids and sugars. Both electrospray-ionization (ESI) (Ge et al. 2001, Schrader et al. 2002) and matrix-assisted laser desorption/ionization (MALDI) (Kang et al. 2000, 2001, Schlüter et al. 2003) mass spectrometry have been used for determination of enzyme activities. Re- cently, we described the use of MALDI-ToF mass spectrometry for the quantification of low molecular weight substrates and products of enzyme reactions and the investigation of enzyme activities (Bungert et al. 2004). A major difficulty in quantification with this method is the inhomogeneity in sample spots which can be overcome by using ionic liquid matrices (ILM) (Zabet-Moghaddam et al. 2004). Here we investigate the use of MALDI-ToF MS as a tool for the screening of enzymes. As model system, ten variants of pyranose oxidase (POx) from the white rot fungi Peniophora sp. converting glucose to the