Pure Appl. Chem., Vol. 75, Nos. 2–3, pp. 309–316, 2003. © 2003 IUPAC 309 Exploitation of proteomics strategies in protein structure–function studies* Gennaro Marino 1,2,‡ , Piero Pucci 1,2 , Leila Birolo 1 , and Margherita Ruoppolo 2,3 1 Dipartimento di Chimica Organica e Biochimica, Facoltà di Scienze Biotecnologiche, Università di Napoli “Federico II”, Napoli, Italy; 2 CEINGE, Biotecnologie Avanzate, scrl, Napoli, Italy; 3 Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli “Federico II”, Napoli, Italy Abstract: Mass spectrometry plays a central role in structural proteomics, particularly in highly intensive structural genomics projects. This review paper reports some examples taken from recent work from the authors’ laboratory and is aimed at showing that modern pro- teomics strategies are instrumental in the integration of structural genomic projects in fields such as: (i) protein–protein interactions, (ii) protein–DNA interactions, (iii) protein–ligand interactions, and (iv) protein-folding intermediates. INTRODUCTION Mass spectrometry (MS) is nowadays the core methodology in structural proteomics [1]. It plays a note- worthy role in highly intensive projects aimed at giving a “form” to the monodimensional inputs of genome sequencing (structural genomics). A number of strategies that combine protein chemistry pro- cedures such as limited proteolysis, selective chemical modifications, and/or hydrogen/deuterium (H/D) exchange with the unsurpassed sensitivity and dynamic range of analysis of MS, may actually provide direct answers to different structural problems such as: (i) probing protein–protein interactions, (ii) probing protein–DNA interactions, (iii) probing protein–ligand interactions, and (iv) probing protein- folding intermediates. Some examples aimed at showing that modern proteomics strategies are instrumental in the inte- gration of structural genomic projects are outlined and discussed in this review paper. PROBING PROTEIN–PROTEIN INTERACTIONS A critical step toward the understanding of protein–protein interactions consists in the structural de- scription of the contact regions within the complex. Focal points of the protein complex “form” may be zoomed-out by the integration of protein chemistry and MS advanced tools. The mono-dimensional data then can eventually be locked on a 3D template by the judicious use of molecular modeling and dynamics. Structural proteomics actually combine the use of traditional protein chemistry tools (i.e., limited proteolysis and chemical reagents) duly revisited, with a modern MS armory. The revisitation essentially consists in spotting only single chemical events occurring on the protein substrate. In order to finely tune the specified reaction to this end, an array of experiments can be monitored by electro- * Pure Appl. Chem. 75, 141–419 (2003). An issue of reviews and research papers based on lectures presented at the 23 rd IUPAC International Symposium on the Chemistry of Natural Products, Florence, Italy, 28 July–2 August 2002 on the theme of natural products. ‡ Corresponding author: Dipartimento di Chimica Organica e Biochimica, Università di Napoli “Federico II”, Complesso Universitario di Monte Sant’Angelo Via Cynthia, 4 80126-Napoli, Italy; Tel: +39-081 674312 or 674476; Fax: +39-081 674313; E-mail: gmarino@unina.it