A two-dimensional gel protein database of noncultured total normal human epidermal keratinocytes: identification of proteins strongly up-regulated in psoriatic epidermis

A two-dimensional (2-D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ-SCAN and PDQUEST software) 2-D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celis et al., Leukemia 1988, 2, 561-602) as well as by 2-D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up-regulated in psoriatic epidermis (Celis et al., FEBS Letters, in press).     

Microsequencing of proteins recorded in human two-dimensional gel protein databases.

Sixty-six human proteins recorded in the master transformed human epithelial amnion cells (AMA) (55) and keratinocyte (11) two-dimensional gel protein databases have been microsequenced since the last publication of the AMA database (Electrophoresis 1990, 12, 989-1071). Coomassie Brilliant Blue stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes and in situ digested with trypsin. The eluting peptides were separated by reversed-phase high performance liquid chromatography (HPLC), collected individually and sequenced. Computer searches using the FASTA and TFASTA programs from the Genetics Computer Group indicated that 29 of the analyzed polypeptides correspond to hitherto unknown proteins.     

A two-dimensional gel database of human plasma proteins.

An updated two-dimensional electrophoretic map of human plasma proteins is presented, together with a complete listing of the individual protein spots, their locations, size and isoelectric points relative to internal charge standards. Forty-nine polypeptide species are identified, many consisting of multiple spots differing in glycosylation or sequence (e.g., immunoglobulins). A further series of 35 as yet uncharacterized proteins is indicated.     

A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

A two-dimensional (2-D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine-labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer-aided 2-D gel electrophoresis. The protein numbers in this database differ from those reported in an earlier version due to changes in the scanning hardware (Celis et al., Electrophoresis 1990, 11, 242-254). Annotation categories reported include: "protein name" (listing 207 known proteins in alphabetical order), "basal cell markers", "differentiation markers", "proteins highly up-regulated in psoriatic skin", "microsequenced proteins" and "human autoantigens". For reference, we have also included 2-D gel (isoelectric focusing) patterns of cultured normal and psoriatic keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2-D gel protein database will be updated yearly in the November issue of Electrophoresis.     

Towards a two-dimensional database of common human proteins.

A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.     

A two-dimensional gel database of rat liver proteins useful in gene regulation and drug effects studies.

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.     

Rat brain proteins: two-dimensional protein database and variations in the expression level

A two-dimensional database of rat brain proteins was constructed. Brain samples from newborn animals were analyzed by two-dimensional electrophoresis and the proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry. The database comprises 210 different proteins, the majority of which are structural components, heat shock proteins and enzymes with various catalytic activities. Several minor differences in the expression level were detected, mainly of quantitative nature, which most likely represent allelic differences. The map may be useful in studies of neurological disorders in animal models of human diseases.     

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.     

The master two-dimensional gel database of human AMA cell proteins: towards linking protein and genome sequence and mapping information (update 1991).

The master two-dimensional gel database of human AMA cells currently lists 3801 cellular and secreted proteins, of which 371 cellular polypeptides (306 IEF; 65 NEPHGE) were added to the master images during the last 10 months. These include: (i) very basic and acidic proteins that do not focus under normal running conditions and (ii) low-abundant proteins that can only be detected after prolonged gel exposure. Annotation categories updated in this version include "protein name", "antibody against protein", "cellular localization", and "microsequenced proteins". New entries include "human autoantigens" and "cDNAs". For convenience we have included an alphabetical list of all known proteins recorded in this database. In the long run, the main goal of this database is to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various cellular functions both under physiological and abnormal conditions.     

Workshop on two-dimensional gel protein databases.

A workshop on two-dimensional gel electrophoresis (2-DE) protein database, organized by the Committee on Data for Science and Technology (CODATA) of the International Council of Scientific Unions Task Group on Biological Macromolecules, was held at the CODATA Secretariat in Paris on March 9, 1992. Eleven scientists from eight different countries represented various aspects of 2-DE analysis--namely, cellular protein database development and protein microsequencing methodologies. The purpose of the workshop was to explore means of integrating the rapidly expanding body of information on 2-DE resolved proteins from different laboratories. A major proposal emanating from the workshop was the establishment of an intermediary or "relational" 2-DE gel protein database. This intermediary database, which would catalogue pertinent information on 2-DE resolved proteins (experimental source, 2-DE loci, biological information, etc.) could be an adjunct to, and accessed through, the existing international protein sequence databanks. It would function as a pointer for researchers to the individual 2-DE protein databases where primary and more specialized 2-DE data would be housed.     

Characterization of human skin fibroblast extracellular proteins by two-dimensional polyacrylamide gel electrophoresis

Human skin fibroblasts secrete over 50 proteins into the culture medium. In this paper these are characterised using two-dimensional polyacrylamide gel electrophoresis and peptide mapping of proteins metabolically labelled in the presence and absence of tunicamycin. Thirty of these proteins have been shown to be N-glycosides, 4 are O-glycosides and 10 are not glycosylated. Of the major proteins, groups 1-4 have previously been shown to be fibroblast specific. Peptide mapping and tunicamycin treatment has identified that groups 1 and 2, and 3 and 4 are closely related and that groups 1 and 3 arise by N-glycosylation of 2 and 4, respectively. The unglycosylated precursor forms of several other proteins have also been identified. This approach to the analysis of protein secretion provides an abundance of information on many proteins simultaneously and can be used to assess the changes in protein secretion associated with development, and to identify extracellular growth factors and other regulatory proteins.     

A human myocardial two-dimensional electrophoresis database: protein characterisation by microsequencing and immunoblotting.

This communication briefly describes how a human heart two-dimensional electrophoresis (2-DE) protein database is being established in our laboratory. The database contains more than 1500 polypeptides and approximately fifty proteins from 2-DE gels of human myocardial tissue have been characterised. Information about the proteins has been compiled including molecular weight (M(r)), isoelectric point (pI), sample spot (SSP) number, protein name, partial sequence, and antibody reacting with the protein. The first stage of this project involves the investigation of protein with pIs in the range pH 4-7. Future studies will employ immobilised pH gradient (IPG) gels as the first dimension of the 2-DE to examine basic proteins. The ultimate goal of this project is to establish a global picture of human heart protein expression in both normal and disease conditions.     

Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins

The human plasma protein patterns obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a good model system for post-translational modifications because of the existence of several "ladders" of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883-906], so-called "trains" of spots. Our investigation of several proteins, amongst others beta2-microglobulin and the haptoglobin chains, found the differences in isoelectric points (p/) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2-D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separated and quantified by reversed-phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine-containing and deamidated asparagine-containing peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2-D gel consists of a mixture of protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the "ladder" is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.     

Identification of proteins from human cerebrospinal fluid, separated by two-dimensional polyacrylamide gel electrophoresis

The aim of this work is to display the protein composition of the cerebrospinal fluid by two-dimensional (2-D) gel electrophoresis and identify it using different mass spectrometric techniques. This will enable us to present an overview of the proteins in human cerebrospinal fluid. The comparison of 2-D gels will help us to analyze the normal protein variability in healthy persons and specific protein variations in patients with different neurological diseases (e.g., morbus Alzheimer, chorea Huntington). However, it is not possible to carry out 2-D gel electrophoresis directly with human cerebrospinal fluid due to the high amount of salts, sugars and lipids present. In addition, the total amount of protein is only as high as 0.3-0.7 microg/microL. Therefore, concentration and desalting steps using precipitation and ultrafiltration are necessary. To date we have been able to identify more than 65 spots from 2-D gels using matrix assisted laser desorption/ionization-mass spectrometry and electrospray ionization-mass spectrometry.     

Pomegranate fruit extract modulates UV-B-mediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa B in normal human epidermal keratinocytes paragraph sign

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B-induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B-mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10-40 microg/mL) for 24 h before UV-B (40 mJ/cm(2)) exposure dose dependently inhibited UV-B-mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 microg/mL) inhibited UV-B-mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B-mediated activation of nuclear factor kappa B (NF-kappaB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated degradation and phosphorylation of IkappaBalpha and activation of IKKalpha. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated nuclear translocation and phosphorylation of NF-kappaB/p65 at Ser(536). Taken together, our data shows that PFE protects against the adverse effects of UV-B radiation by inhibiting UV-B-induced modulations of NF-kappaB and MAPK pathways and provides a molecular basis for the photochemopreventive effects of PFE.     

Sequence analysis of proteins separated by polyacrylamide gel electrophoresis: towards an integrated protein database

Improved technologies or the synergistic use of complementary methods enhance the efficiency of research and permit the exploration of new approaches for the investigation of complex problems. High sensitivity protein sequence analysis and polyacrylamide gel electrophoresis are such complementary methods. Here we summarize the current status of high sensitivity sequence analysis of proteins separated in polyacrylamide gels and discuss strategies by which this technology can enhance biological research by generating new approaches for the solution of complex, multifacetted problems. Finally, we outline imminent technological advances in the area of high sensitivity protein sequence analysis and argue that further technological developments will ultimately lead to the generation of an integrated protein database (containing structural and functional as well as physiological information in an easily accessible form) of all the proteins separated by high resolution two-dimensional gel electrophoresis.     

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.