Giovanni Candiano 1 Luca Musante 1 Maurizio Bruschi 1 Gian Marco Ghiggeri 1 Ben Herbert 2 Francesca Antonucci 3 Pier Giorgio Righetti 3 1 Nephrology Section, G. Gaslini Children’s Hospital, Genova, Italy 2 Proteome Systems, North Ryde, Sydney, NSW, Australia 3 Department of Agricultural and Industrial Biotechnologies, University of Verona, Verona, Italy Two-dimensional maps in soft immobilized pH gradient gels: A new approach to the proteome of the Third Millennium Same major improvements in proteome analysis of cytosolic and membrane proteins by two-dimensional mapping are here reported. A much improved transfer of proteins from the first to the second dimensional sodium dodecyl sulfate (SDS)-gel is obtained by simply diluting the gel matrix, normally composed of 4%T polyacrylamide in all commercially available Immobiline strips down to as low as 3%T. In the analysis of total lysates of platelets, this augmented transfer has been evaluated as being 2–3 times higher than in standard 4%T gels. A second major improvement, in the case of analysis of membrane protein preparations, has been demonstrated to consist in a delipidation step in a tertiary solvent mixture composed of tri-n-butyl phosphate:acetone:methanol in a 1:12:1 ratio. By adopting this protocol, large amounts of spectrins (240–220 kDa, filamentous proteins of the red blood cell membranes) could be transferred vs. essen- tially none when delipidation was omitted. The present report also confirms the impor- tance of a reduction and alkylation step of the protein sample prior to all electrophore- tic steps, including focusing in the Immobiline gel, as recently reported by Herbert etal. (Electrophoresis 2001, 22, 2046–2057). Keywords: Two-dimensional maps / Porous immobilized pH gradient gels / Platelets / Red blood cells EL 4768 1 Introduction At the start of the Third Millennium, two-dimensional (2-D) maps, as epitomized by a focusing step in immobilized pH gradients (IPGs), followed by an orthogonal SDS- PAGE step, have been adopted as a well-ingrained and well-standardized technique [1]. Commercial IPG strips are now offered by several companies; SDS-PAGE slabs, at constant %Tor as porosity gradients, can also be pur- chased and even kits for silver staining are now commer- cially available. Although it would thus appear that no new developments could possibly be foreseen, some impor- tant new methodologies have in fact opened a new obser- vation window in the proteome of the Third Millennium. Already at the end of the last millennium it was recognized that conventional 2-D maps, as routinely performed, could not possibly match the complexity of the proteome of even a single cell population [2]. This called for pre frac- tionation techniques, which were developed on different chromatographic platforms by the Fountoulakis group [3–6]. A refinement of this step, consisting on harvesting isoelectric fractions from a multicompartment electrolyzer with isoelectric membranes, fully compatible with the IPG dimension, was proposed at the beginning of the new mil- lennium, and proved to be a remarkable step ahead in increasing the depth of field in deciphering the proteome [7]. Another important development, curiously ignored up to the present, has been a reduction and alkylation step as a fundamental sample treatment prior to any electro- phoretic fractionation, including the first IPG dimension [8–10]. This proved to be highly beneficial for at least two reasons: (i) it prevented formation of spurious spots, due to oligomers among like and unlike polypeptide chains; (ii) it permitted transfer of substantially higher amounts of proteins from the IPG strip into the SDS-laden gel slab. Although there could possibly be room for other, presently unforeseen, methodological developments, there is still at least one other issue to be addressed, namely the optimi- zation of the matrix porosity in the first dimensional, IPG strip. Although in all possible IPG recipes today published a polyacrylamide matrix consisting of 4%T seems to have been universally adopted, we have wondered if other gel formulations, consisting in casting softer gels, could per- mit better transfer of polypeptide chains from the first dimension in the final SDS-slab surface, ultimately dis- playing the 2-D image. Many authors have lamented, e.g., poor transfer of large proteins trapped in the first dimensional IPG gel [11]. We report here the results of such an investigation, coupled to improved delipidization protocols for analysis of membrane proteins. Correspondence: Prof. P. G. Righetti, Department of Agricultural and Industrial Biotechnologies, University of Verona, Strada le Grazie No. 15, I-37134 Verona, Italy E-mail: righetti@sci.univr.it Fax: +39–45–8027901 Abbreviations: DTE, dithioerythritol; RBC, red blood cell; TBP , tributylphosphine 292 Electrophoresis 2002, 23, 292–297 ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 0173-0835/02/0201–292 $17.50+.50/0