© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 73 Biotechnol. J. 2009, 4, 73–87 DOI 10.1002/biot.200800250 www.biotechnology-journal.com 1 Introduction The biological activity of proteins is determined, besides by their covalent structure, by events that affect their conformation and ability to interact with other molecules. Conformational transitions, propensity to unfold and/or aggregate in cellular or artificial environments, as well as binding to other proteins, ligands, cofactors and inhibitors, are all is- sues of paramount importance when analyzing the properties of native or recombinant proteins. In- formation about such properties is useful to opti- mize conditions for protein employment or to de- sign strategies of improvement. Protein conforma- tional stability and dynamics are also crucial for the mode of action of protein-based drugs or for their stability both during storage and after administra- tion to the patient [1, 2]. On the side of industrial biotechnology, characterization of the conforma- tional stability of enzymes towards challenging op- erational conditions or conformational changes in- duced by storage or addition of stabilizers might provide a body of information useful in the imple- mentation of biocatalytic processes. A number of biophysical techniques contribute to our capability to investigate protein structural properties in solution. Among them, circular dichroism [3], fluorescence [4] and nuclear mag- netic resonance [5] are routinely employed. This review article is aimed at drawing attention to the potential impact of electrospray-ionization mass spectrometry (ESI-MS) in biotechnology,by the use of a few examples from the work of our and other laboratories. After an introduction to the theory and to the main technical aspects of the method, we will focus on three main issues: conformational Review Electrospray-ionization mass spectrometry as a tool for fast screening of protein structural properties Rita Grandori, Carlo Santambrogio, Stefania Brocca, Gaetano Invernizzi and Marina Lotti Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy Since the early 1990s, electrospray-ionization mass spectrometry (ESI-MS) has encountered grow- ing interest as a complementary tool to established biochemical and biophysical methods for in- vestigating protein structure and conformation. Nowadays, applications of ESI-MS to protein in- vestigation span from the area of analytical biochemistry to that of structural biology. This review focuses on applications of this technique to the analysis of protein conformational properties and molecular interactions, underscoring their possible relevance for molecular biotechnology, al- though representing a still very young field. An introductive section presents the major issues re- lated to theoretical and technical aspects of ESI-MS under non-denaturing conditions. Examples from our work and from the literature illustrate which kind of information can be obtained con- cerning key issues in biotechnology such as stability and aggregation of proteins under both near- native and challenging conditions, and interactions with other proteins, ligands and cofactors. Keywords: Electrospray-ionization mass spectrometry · Protein conformation · Protein-ligand interactions · Protein-protein interactions Correspondence: Prof. Marina Lotti, Dipartimento di Biotecnologie e Bio- scienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy E-mail: marina.lotti@unimib.it Fax: +39-02-64483565 Abbreviations: BLG, bovine lactoglobulin; CSD, charge-state distribution; ESI, electrospray ionization; TFE, trifluoroethanol; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; PLG, porcine lac- toglobulin; RXR, retinoid X receptor; TOF, time-of-flight; VAO, vanillyl-alco- hol oxidase Received 1 October 2008 Revised 13 November 2008 Accepted 3 December 2008