Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2-D LC-MS/MS) technique has high capability of resolving peptides, and is used for analysis of tryptic peptides derived from highly complex protein mixtures such as plasma. However, the detection of low-abundant proteins and low-molecular-weight proteins is often very difficult in this system, because major peptides from high-abundant proteins such as albumin mostly disturb the separation of minor peptides from low-abundant proteins. To resolve these problems, different methods of sample preparation for in-solution trypsin digestion were tested, and the optimized method was applied to the 2-D LC-MS/MS analysis of proteins secreted from human hepatoma cell line, HepG2. We could identify 247 and 142 proteins from the chemically and thermally denatured samples, respectively. Among them, 71 proteins were identified in common to both methods, while most of the proteins were identified using either of the two procedures. In addition to these denaturation methods, the molecular-mass cutoff via ultrafiltration improved the efficiency in identifying low-molecular-weight proteins. Finally, we could identify 478 secreted proteins in total using the combination of these processes. These data indicate that in sample preparation the combination of various denaturation methods, as well as molecular-mass cutoff, are very critical for the identification of a wider range of low-abundant proteins via 2-D LC-MS/MS analysis.

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The diverse functions of the kidney are fundamentally fulfilled by the nephron, the functional unit of the kidney. The nephron consists of several functionally and structurally different segments, which spans the kidney from the cortex through the medulla. The functional specialization along the nephron and specific localization of the different segments should be reflected in the protein expression profiles of distinct anatomical compartments of the kidney. We performed a two-dimensional gel electrophoresis (2-DE)-based, quantitative, proteomic analysis to identify proteins differentially expressed in the cortex, medulla, and glomeruli purified from cortex of normal human kidney. The protein extracts of the three compartments obtained from 4 normal subjects were separated on 2-DE gels, and analyzed by using a 2-DE gel image analysis software. We detected 1,810 valid protein spots in the glomerulus, 1,758 in the cortex, and 1,275 spots in the medulla. Statistical analysis by one-way ANOVA followed by Tukey's post-hoc test revealed that 616 protein spots were differentially expressed in the glomeruli, 582 in the cortex, and 469 in the medulla. Among these protein spots, spots expressing in considerably higher relative amounts in one of the three compartments were selected and subjected to identification by peptide mass fingerprinting method with a MALDI-TOF mass spectrometer, which led to identification 19 protein spots preferentially expressed in the glomerulus, 35 in the cortex, and 21 in the medulla. The distinct protein expression profiles in different kidney compartments may serve as a resource for further study of physiology and pathophysiology of the human kidney.

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We developed a simple method for purifying peptides from crude extracts of animal tissues. Peptides free of high-molecular-mass (HMM) proteins were prepared by differential solubilization of proteins and peptides followed by tricine-SDS-PAGE. Proteins and peptides in a tissue extract were co-precipitated by slowly dropping the extracts into acetone at −40°C. The precipitates were dissolved in 2 mM DTT and separated by centrifugation. The peptide-enriched supernatant was resolved by tricine-SDS-PAGE and portions of the lanes below 10 kDa were excised. Peptides were extracted from the gel pieces in 40% acetonitrile and fractionated by two-step reversed-phase HPLC. We evaluated the method by extracting peptides from the livers and kidneys of rats and diabetic model mice. The HPLC elution profiles of peptide mixtures were reproducible, peptide recovery from tricine-SDS-PAGE gels was high and we identified one HPLC-fractionated peptide by amino-acid sequencing. The present method should serve as a good foundation for future peptidomics.

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Comparing protein expression in the cerebrospinal fluid (CSF) of rheumatoid arthritis (RA) patients with that of controls, makes possible the uncovering of proteins that affect disease progression and regulate responsiveness to drugs. Two-dimensional gel electrophoresis (2-DE) and silver staining were used for identifying disease-associated CSF proteins in RA patients. First, to enhance the detection of CSF proteins and to improve the separation of their isoforms by 2-DE, CSF samples were pre-treated with an albumin and IgG removal kit, then by acetone precipitation. The 2-DE analysis revealed more than 1600 spots by the removal of albumin and immunoglobulin from CSF. The expression of the protein spots was not greatly changed in either group, but some notable changes in protein spots were observed in two RA samples. In particular, the expression of an approximately 50 kD protein increased markedly, whereas that of two sequential protein spots of 10-15 kD and with neutral pI decreased in the RA samples. These preliminary results suggest that the proteomic method is conducive to clarifying the mechanism of RA crises, and that some of the expression-changed proteins may be new candidates for disease-associated proteins of RA.

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Peptides play a central role as reaction specific probes for screening protein libraries for target enzymes with protein-modifying activities with the automated platform technology named mass spectrometry assisted enzyme screening (MES). The protein libraries are generated by fractionating protein extracts and by immobilizing the proteins covalently to activated affinity beads. The innovative idea of the MES-system is to incubate the fractions of the protein library with special designed peptides that serve as substrates for protein modifying enzymes and to detect the resulting reaction products by mass spectrometry, if the target enzyme is present in a fraction of the protein library. The peptide sequences, selected for the reaction specific probes, are usually parts of the endogenous protein substrates. The main advantage of the MES approach is that even complex protein mixtures can be screened for enzymatic activities. The MES technique is suited for the search for unknown target enzymes with defined catalytic reaction profiles, for investigating the metabolism of defined substrates in cells or tissues and for comparing defined enzyme activities in organisms in different states. Therefore the application of the MES system includes a wide area, from the identification of new drug targets to the identification of enzymes relevant for biotechnological processes. Here the MES system is demonstrated for the screening for urotensin-II (U-II) metabolising enzymes in renal tissue. With MES des-Val-U-II was determined as major metabolite of U-II of renal tissue proteins.

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We developed a collaborative proteomics framework with XML-based 2DPAGE database and XML viewer optimized for the proteome database. Prior to the development of the framework, we optimized the XML format of our TMIG-XML-2DPAGE database for efficient data sharing in a collaborative research community. The data format is comprised with 2 layers of 2-D gel map and spot protein information. The function of clickable imagemap for representing spot protein information is available both in the proteome data management system with PHP and in the XML viewer with Object Pascal. Individual spots on an experimental 2-D gel image are easily matched to corresponding spots on a standard 2-D gel map in the TMIG-XML-2DPAGE database using the function of the XML viewer.

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