Proteomic analysis of the rat liverIntroductionExperimentalMaterialsSample preparationTwo-dimensional gel electrophoresisMatrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS)ResultsTwo-dimensional electrophoretic analysisIdentity assignmentProtein abundance and functionSubcellular locationHydrophobicityHeterogeneityFrequency of detectionDiscussionAcknowledgementsReferencesJournal of Chromatography B, 782 (2002) 197–218www.elsevier.com/locate/chrombP roteomic analysis of the rat livera, b*Michael Fountoulakis , Laura SuteraRoche Center for Medical Genomics Inc.,F.Hoffmann-La Roche Ltd.,Building93-444, 4070Basel,SwitzerlandbPharmaceutical Research,Drug Safety,Basel,SwitzerlandAbstractRat is a useful, widely used animal model for biological and toxicity studies. We analyzed total and cytosolic rat liverproteins by applying proteomics technologies. The proteins were separated by two-dimensional electrophoresis employingbroad and narrow range immobilized pH gradient strips, followed by MALDI-MS analysis of the tryptic digests. Twohundred and seventy-three different gene products were identified, of which approximately 60% were enzymes with a broadspectrum of catalytic activities. Most of the identified proteins were detected in other rat protein samples as well, which wereanalyzed 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 10spots corresponded to one gene product. The database includes a large number of proteins known to be involved intoxicology-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 liver1 . Introduction the last few years and it now allows the detectionand mapping of all species of a proteome, which areProteomics is the high-throughput, large-scale, expressed in sufficient amounts to be detected in amainly automated analysis of protein mixtures. two-dimensional (2-D) gel. Many laboratories, ourProteomic analysis is a useful tool in investigating own included, have undertaken the task to find andbiological events, as it provides us with significant map the proteins of various proteomes. Thus, todayinformation about the particular proteome, i.e., which many 2-D databases include several hundreds ofare the abundant gene products and how their levels different gene products [3–11]. The 2-D electro-and modifications change in response to the effect of phoretic analysis has certain limitations, concerningvarious internal or external factors, such as diseases, the detection of hydrophobic proteins and proteinstoxic agents and environmental changes. Moreover, with extreme size and charge values [2,10–12].it facilitates protein–protein interaction and protein Other emerging proteomics technologies not relyingstructure studies [1,2]. The sensitivity of the on 2-D gels appear to detect a higher number of geneproteomics technologies has been largely improved products, but they are compromised by quantificationweaknesses [13,14]. However, there is still a largediscrepancy between possible and detected gene*Corresponding author. Tel.: 141-61-688-2809; fax: 141-61-products in a proteome. To increase the likelihood of691-9391.detection of low-abundance proteins in complexE-mail address:[email protected] (M. Foun-toulakis). biological mixtures, a proteomic analysis should be1570-0232/02/$ – see front matter  2002 Elsevier Science B.V. All rights reserved.PII: S1570-0232(02)00562-7198 M.Fountoulakis,L.Suter / J.Chromatogr.B782 (2002) 197–218directed to simpler protein fractions, each containing2 .2.Sample preparationa lower number of components in comparison withthe starting material. The separation of the protein Animals were sacrificed using CO . Livers from2mixture into organelle fractions prior to the 2-D two control animals were flushed through the hepaticelectrophoretic analysis is usually the first step to vein with a cold NaCl solution to eliminate excessiveincrease the probability of detecting low-copy-num- blood content. For preparation of the total proteinber gene products [12]. extract, liver tissue (1.0 g) was suspended in 10 mlOne of the most frequent applications of of sample buffer consisting of 20 mM Tris, 7 Mproteomics is the investigation of toxic events, as it urea, 2 M thiourea, 4% CHAPS, 10 mM 1,4-enables the efficient generation of toxicity-related dithioerythritol, 1 mM EDTA and a mixture ofprotein patterns, which may be useful in predicting protease inhibitors (1 mM PMSF and one tablettoxicity of drug candidates [15,16]. We have applied complete姠 (Boehringer Mannheim) per 50 ml ofproteomics technologies to study changes in the suspension buffer) and phosphatase inhibitors (0.2levels of liver proteins of mice treated with acet- mM Na VO and 1 mM NaF). The suspension was23aminophen [9], of rats treated with carbon tetra- homogenized with the use of a Polytron homogenizerchloride, as well as changes of brain proteins of rats (Kinematica, Luzern, Switzerland) for approximatelytreated with the neurotoxin kainic acid, a cyclic 1 min, sonicated for 30 s and centrifuged atanalogue of glutamate [17,18]. In all cases, the 150 000 g for 45 min. The supernatant contained thedifferential protein expression studies revealed the total liver proteins solubilized in the IEF-compatiblepresence of significant derangements in the levels of agents.a series of protein classes, following administration For the preparation of the cytosolic fraction, liverof the toxic agents. To facilitate the performance of tissue (1.0 g) was suspended in 10 ml of 20 mMtoxicity studies and the investigation of animal Hepes–OH, pH 7.5, containing 250 mM sucrose, 1models of human diseases, we constructed two-di- mM EDTA, 5 mM dithioerythritol and protease andmensional databases for mouse liver total [9], cyto- phosphatase inhibitors as above. The suspension wassolic and microsomal proteins [10], as well as for rat homogenized with the use of a PTFE/potterbrain total proteins [7]. In a previous study, we homogenizer and centrifuged at 800 g for 10 min toanalyzed the rat liver mitochondrial proteins [19]. remove nuclei and undissolved material. The super-Here we performed a large-scale proteomic analysis natant was centrifuged at 10 000 g