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Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulator in cholesterol biosynthesis and HMG CoA reductase inhibitors (statins) have become a widely prescribed family of lipid lowering agents. Cholesterol synthesis occurs predominantly in liver which is the target organ of statins. We studied the effects of fluvastatin (Lescol®), a member of the statin family, on hepatic protein regulation. Male F344 rats treated with 0.8 mg/kg per day fluvastatin or 24 mg/kg per day fluvastatin for 7 days showed treatment-related changes in 58 liver proteins (P

Despite the widespread use of cyclosporine A (CsA), its mechanism of action and side effects are not yet completely understood. There exists a large body of evidence suggesting that disturbance of calcium homeostasis is a critical step in the cascade of cellular and molecular events induced by the drug. As recently shown in our laboratory by two-dimensional protein gel electrophoresis (2-DE) analysis of kidney homogenates, CsA induced numerous changes in several kidney proteins. One kidney protein in particular was shown to be strongly down-regulated by the drug. In this work we report the identification of the strongly decreased kidney protein as calbindin-D 28kDa, a vitamin D-dependent calcium-binding protein associated with calcium handling by cells. The assignment of the down-regulated protein spot is based on its internal amino acid sequence analysis and its specific reaction with a monoclonal antibody raised against calbindin-D 28kDa. In kidney homogenates of male Wistar rats treated with 50 mg/kg/d CsA for up to 28 days, calbindin levels were measured by ELISA and were shown to be continuously decreased with prolonged CsA treatment. To our knowledge, this is the first report describing the effect of CsA on kidney calbindin-D 28kDa protein levels. Further studies are needed to elucidate whether the CsA-mediated down-regulation of the calcium-binding protein calbindin-D 28kDa may be a critical factor for the renal adverse effects induced by this drug.

The liver is composed of a variety of cells that form a functional unit involved in uptake, synthesis, metabolism, and secretion. Until recently, most studies examining liver function did not analyze the specific proteins expressed or functions performed by the multiple individual cell types that constitute the hepatic mass. In the last decade, novel isolation methods have been developed that allow the purification of liver cell populations highly enriched in one type of liver cell. Here, we present a detailed two-dimensional (2-D) protein map of rat bile duct epithelial cells (i.e., cholangiocytes) using a recently developed isolation procedure. In addition, we identify 27 major cholangiocyte proteins either by comparison to maps of known rat liver proteins (based on pI and Mr) or by tryptic digestion and microsequencing. Finally, we compare the relative abundance of individual proteins present in cholangiocytes to whole liver as well as hepatocyte-specific proteins. Our results show that cholangiocytes express a unique array of individual proteins. The cholangiocyte 2-D protein pattern is markedly different from that of isolated rat hepatocytes or whole rat liver, with high levels of proteins previously known to be expressed by cholangiocytes (e.g., cytokeratins, actins) as well as protein not previously demonstrated to be expressed at high levels (e.g., annexin V, selenium binding protein). We conclude that this cholangiocyte-derived, 2-D protein map will be a crucial resource for studies directed at our understanding of cholangiocyte physiology and pathobiology.

Proteins implement most biological functions at the molecular level. As one might expect based on this fact, it appears that the altered functional states associated with toxic effects involve changes in the abundance or structure of proteins. Although numerous specific assays exist to measure changes in the abundance of individual proteins, practical limitations have prevented widespread use of multiple protein assays for the global characterization of toxicity. Recent developments in protein analytical technology are rapidly changing this picture. Two-dimensional gel electrophoresis, a technique capable of resolving and quantitating hundreds of proteins simultaneously, is becoming an automated, high-throughput tool. In parallel, techniques have been developed that allow the resulting deluge of protein measurements to be organized into a prototype Molecular Effects Database™ describing xenobiotic effects in rodent liver. This database can detect, classify, and characterize a broad range of liver toxicity mechanisms. It currently contains approximately 10 million protein measurements, including data on the liver effects of 43 compounds, with a further 50 compounds to be added in 1995. Observed effects range from very broad (sex steroids alter levels of 45% of all liver proteins) to very specific (e.g., hepatic hydroxymethyl glutaryl coenzyme A reductase inhibitors). Companion 2-dimensional databases describing rodent brain and kidney have been initiated, as have linkages to the genomic sequence databases. Assimilation of this approach into research and regulatory toxicology poses an interesting challenge—one that is likely to lead to a radically more sophisticated understanding of toxicity and its biological basis.

We have improved upon the reference two-dimensional (2-D) electrophoretic map of rat liver proteins originally published in 1991 (N. L. Anderson et al., Electrophoresis 1991, 12, 907-930). A total of 53 proteins (102 spots) are now identified, many by microsequencing. In most cases, spots cut from wet, Coomassie Blue stained 2-D gels were submitted to internal tryptic digestion [2], and individual peptides, separated by high-performance liquid chromatography (HPLC), were sequenced using a Perkin-Elmer 477A sequenator. Additional spots were identified using specific antibodies.

A standard two-dimensional (2-D) protein map of Fischer 344 rat liver (F344MST3) is presented, with a tabular listing of more than 1200 protein species. Sodium dodecyl sulfate (SDS) molecular mass and isoelectric point have been established, based on positions of numerous internal standards. This map has been used to connect and compare hundreds of 2-D gels of rat liver samples from a variety of studies, and forms the nucleus of an expanding database describing rat liver proteins and their regulation by various drugs and toxic agents. An example of such a study, involving regulation of cholesterol synthesis by cholesterol-lowering drugs and a high-cholesterol diet, is presented. Since the map has been obtained with a widely used and highly reproducible 2-D gel system (the Iso-Dalt system), it can be directly related to an expanding body of work in other laboratories.