Journal of Chromatography B 782 2002 197 218 www elsevier com locate chromb Proteomic analysis of the rat liver a b Michael Fountoulakis Laura Suter a Roche Center for Medical Genomics Inc F Hoffmann La Roche Ltd Building 93 444 4070 Basel Switzerland b Pharmaceutical Research Drug Safety Basel Switzerland Abstract Rat is a useful widely used animal model for biological and toxicity studies We analyzed total and cytosolic rat liver proteins by applying proteomics technologies The proteins were separated by two dimensional electrophoresis employing broad and narrow range immobilized pH gradient strips followed by MALDI MS analysis of the tryptic digests Two hundred and seventy three different gene products were identified of which approximately 60 were enzymes with a broad spectrum of catalytic activities Most of the identified proteins were detected in other rat protein samples as well which were analyzed in our laboratory Fifteen gene products were detected for the first time These were represented by one spot each whereas most of the frequently detected proteins were represented by multiple spots In average approximately five to 10 spots corresponded to one gene product The database includes a large number of proteins known to be involved in toxicology relevant pathways and may be useful in toxicity prediction studies 2002 Elsevier Science B V All rights reserved Keywords Proteomics Two dimensional database Proteins rat liver 1 Introduction Proteomics is the high throughput large scale mainly automated analysis of protein mixtures Proteomic analysis is a useful tool in investigating biological events as it provides us with significant information about the particular proteome i e which are the abundant gene products and how their levels and modifications change in response to the effect of various internal or external factors such as diseases toxic agents and environmental changes Moreover it facilitates protein protein interaction and protein structure studies 1 2 The sensitivity of the proteomics technologies has been largely improved Corresponding author Tel 141 61 688 2809 fax 141 61691 9391 E mail address michael fountoulakis roche com M Fountoulakis the last few years and it now allows the detection and mapping of all species of a proteome which are expressed in sufficient amounts to be detected in a two dimensional 2 D gel Many laboratories our own included have undertaken the task to find and map the proteins of various proteomes Thus today many 2 D databases include several hundreds of different gene products 3 11 The 2 D electrophoretic analysis has certain limitations concerning the detection of hydrophobic proteins and proteins with extreme size and charge values 2 10 12 Other emerging proteomics technologies not relying on 2 D gels appear to detect a higher number of gene products but they are compromised by quantification weaknesses 13 14 However there is still a large discrepancy between possible and detected gene products in a proteome To increase the likelihood of detection of low abundance proteins in complex biological mixtures a proteomic analysis should be 1570 0232 02 see front matter 2002 Elsevier Science B V All rights reserved PII S1570 0232 02 00562 7 198 M Fountoulakis L Suter J Chromatogr B 782 2002 197 218 directed to simpler protein fractions each containing a lower number of components in comparison with the starting material The separation of the protein mixture into organelle fractions prior to the 2 D electrophoretic analysis is usually the first step to increase the probability of detecting low copy number gene products 12 One of the most frequent applications of proteomics is the investigation of toxic events as it enables the efficient generation of toxicity related protein patterns which may be useful in predicting toxicity of drug candidates 15 16 We have applied proteomics technologies to study changes in the levels of liver proteins of mice treated with acetaminophen 9 of rats treated with carbon tetrachloride as well as changes of brain proteins of rats treated with the neurotoxin kainic acid a cyclic analogue of glutamate 17 18 In all cases the differential protein expression studies revealed the presence of significant derangements in the levels of a series of protein classes following administration of the toxic agents To facilitate the performance of toxicity studies and the investigation of animal models of human diseases we constructed two dimensional databases for mouse liver total 9 cytosolic and microsomal proteins 10 as well as for rat brain total proteins 7 In a previous study we analyzed the rat liver mitochondrial proteins 19 Here we performed a large scale proteomic analysis of total and cytosolic rat liver proteins and identified 273 different gene products 2 Experimental 2 1 Materials Immobilized pH gradient IPG strips were purchased from Amersham Pharmacia Biotechnology Uppsala Sweden Acrylamide was obtained from Serva Heidelberg Germany and the other reagents for the polyacrylamide gel preparation were from Bio Rad Hercules CA USA Ampholytes Resolyte 3 5 10 were purchased from BDH Laboratory Supplies Poole UK CHAPS and thiourea were from Sigma St Louis MO USA urea dithioerythritol and EDTA were obtained from Merck Darmstadt Germany Adult male Wistar rats were purchased from BRL Fullingsdorf Switzerland 2 2 Sample preparation Animals were sacrificed using CO 2 Livers from two control animals were flushed through the hepatic vein with a cold NaCl solution to eliminate excessive blood content For preparation of the total protein extract liver tissue 1 0 g was suspended in 10 ml of sample buffer consisting of 20 mM Tris 7 M urea 2 M thiourea 4 CHAPS 10 mM 1 4dithioerythritol 1 mM EDTA and a mixture of protease inhibitors 1 mM PMSF and one tablet complete Boehringer Mannheim per 50 ml of suspension buffer and phosphatase inhibitors 0 2 mM Na 2 VO 3 and 1 mM NaF The suspension was homogenized with the use of a Polytron homogenizer Kinematica Luzern Switzerland for approximately 1 min sonicated for 30 s and centrifuged at 150 000 g for 45 min The supernatant contained the total liver proteins solubilized in the IEF compatible agents For the preparation of the cytosolic fraction liver tissue 1 0 g was suspended in 10 ml of 20 mM Hepes OH pH 7 5 containing 250 mM sucrose 1 mM EDTA 5 mM dithioerythritol and protease and phosphatase inhibitors as above The suspension was homogenized with the use of a PTFE potter homogenizer and