Behrouz Vaziri 1 Mehdi Rahimpour 2 Naser Eslami 3 Ahmad Fayaz 3 Heshmat Rahimian 2 1 Protein Chemistry Unit, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 2 University of Mazandaran, Babolsar, Iran 3 Rabies Department, Pasteur Institute of Iran, Tehran, Iran Original Paper RP-HPLC prefractionation and its application in expressional proteomics analysis of an in vitro viral infection model Prefractionation of complex protein mixtures is an efficient method for increasing the separation power of 2-DE. RP-HPLC has been successfully utilized as a prefractio- nation method prior to 2-DE. Here we describe the optimization of an efficient RP- HPLC method for prefractionation of baby hamster kidney cell solubilized proteins. A step gradient elution of acetonitrile was optimized and collected fractions were further examined by SDS-PAGE and 2-DE. By utilizing this method an effective increase in separation power of 2-DE is accomplished. Moreover, we describe the application of this method to expressional proteome analysis of a virally infected cell model. Keywords: 2-Dimensional electrophoresis / Prefractionation / Proteomics / Rabies virus / Received: April 8, 2006; revised: May 24, 2006; accepted: May 25, 2006 DOI 10.1002/jssc.200600140 1 Introduction Obtaining an overall proteome picture for a given sam- ple is a challenge in present-day proteome analysis. 2-DE is currently the most widespread method for separation and expressional profiling of complex protein mixtures [1, 2]. Relatively high resolution power in a single separa- tion run, high reproducibility, and compatibility with mass spectrometry are the main advantages of 2-DE [3–5]. However, 2-DE does have some limitations for separating and detecting strongly acidic and basic, very low and high molecular mass, highly hydrophobic, and low copy number proteins [6–8]. Among these limitations, the inability of 2-DE to visualize very low abundance proteins in the presence of highly expressed proteins is a major disadvantage [9]. Different strategies were considered for solving this problem, including various fractionation techniques prior to 2-DE [10–12]. Prefractionation increases not only the separation power of 2-DE but also the chance of detecting the low abundance proteins [13]. Here we report the optimization of a previously described RP-HPLC method [14] for separating the pro- teins in a complex mammalian cell. We describe how the utilization of a prefractionation method, based on the hydrophobic properties of proteins, as an extra dimen- sion prior to 2-DE could affect the resolution power. The efficiency of this method in expressional proteome anal- ysis of baby hamster kidney cells infected by rabies virus is also discussed 2 Experimental 2.1 Chemicals and materials The baby hamster kidney cell line (BHK21-2P) was kindly provided by the cell bank of the Pasteur Institute of Iran. Challenge virus strain (CVS) rabies virus was used for infecting the BHK cells. 2.2 Cell culture and sample preparation BHK21-2P cells were cultured at 378C in minimum essen- tial medium supplemented with 10% v/v fetal serum and 40 mg/mL gentamicin. BHK21-2P cells were inoculated with CVS of rabies at an input multiplicity of infection of 1 LD 50 per 10 cells. Cells were harvested by centrifugation at 10006g and washed three times with elution buffer containing 10 mM Tris (pH 7) and 250 mM sucrose. BHK cells (3610 8 ) were lysed in 1 mL buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10, 50 mM DTT, 40 mM Tris, and 0.2% ampholytes at pH 3–10. The lysate was clarified by centrifugation at room temperature (13 000 rpm, 10 min). The protein concentration was determined by Bradford assay using BSA standards. The lysate was either used immediately or aliquoted and kept at –708C for further use. Correspondence: Dr. Behrouz Vaziri, Pasteur Institute of Iran, 69, Pasteur Ave., Tehran-13164, Iran E-mail: Behrouz-vaziri@pasteur.ac.ir Fax: 98-21-66480780 Abbreviations: BHK, baby hamster kidney; CVS, challenge virus strain. i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 2284 B. Vaziri et al. J. Sep. Sci. 2006, 29, 2284 – 2291