Systematic approach to studying proteins from human platelets. Two-dimensional mapping]

Total proteins of human platelets and their membranes were studied by two-dimensional electrophoresis using the lysis in sodium dodecyl sulfate solution. Analysis of platelets from 30 donors revealed the presence of 105 repeating fractions on the electrophoregrams. A two-dimensional map of platelet proteins in the molecular mass versus relative electrophoretic mobility plot was constructed. This map made it possible to localize membrane proteins and albumin as well as a protein immunologically related to phenylalanine hydroxylase. Electrophoretic variants of 13 platelet polypeptides were identified.     

Two-dimensional map of membrane proteins from human erythrocytes

Human erythrocyte membrane proteins were analyzed by a modified two-dimensional electrophoresis performed according to O'Farrell. This method was used to construct a two-dimensional map of human erythrocyte membrane proteins. The map plotted in the coordinates "relative molecular mass versus relative electrophoretic mobility during IEF" was used for the characterization of 189 proteins. The position of major membrane proteins in the map was determined on the basis of their Mr, pI as well as literature data. Carboanhydrase was positioned by coelectrophoresis. A comparative analysis of erythrocyte membrane and cytosol preparations by two-dimensional protein mapping revealed that some of erythrocyte proteins have dual localization.     

Two-dimensional mapping by the high-performance liquid chromatography of oligosaccharides released from glycosphingolipids by endoglycoceramidase

A two-dimensional sugar mapping method has been developed by which sensitive, reproducible, and simple analysis can be carried out on the structures and compositions of oligosaccharides released from glycosphingolipids by endoglycoceramidase. The oligosaccharides were labeled quantitatively with an ultraviolet-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-oligosaccharides were separated first on an amide-silica column and then on a C4-silica column by high-performance liquid chromatography. The acidic ABEE-oligosaccharides were eluted as a group at the start of the chromatography while the neutral ABEE-oligosaccharides were separated according to size and structure on an amide-silica column using an eluent without salt. The acidic oligosaccharides were separated according to size and structure when rechromatographed on the same column using an eluent containing KH2PO4. NeuAc-containing ABEE-oligosaccharides were extensively separated from the corresponding NeuGc derivatives. The ABEE-oligosaccharides separated on an amide-silica column were then chromatographed on a column of C4-silica on which lactotriose and neolacto-series oligosaccharides were clearly shown to be separated from the others. On the basis of the retention times of the individual ABEE-oligosaccharides on two separate columns, 9 neutral and 15 acidic oligosaccharides derived from glycosphingolipid standards were two-dimensionally mapped without overlapping. The gangliosides of a human chondrosarcoma tissue and glycosphingolipids of tumor tissue of FBJ virus-transformed murine osteosarcoma cells were analyzed by this method in conjunction with exoglycosidase treatment. At least 11 species of glycosphingolipids were identified in both cases.     

Direct and simple detection of recombinant proteins from cell lysates using differential scanning fluorimetry

A simple, inexpensive, and universal method to quantify the recombinant proteins in Escherichia coli cell lysate using differential scanning fluorimetry (DSF) is reported. This method is based on the precise correlation between Δ(fluorescence intensity) determined by DSF and the amount of protein in solution. We first demonstrated the effectiveness of the DSF method using two commercially available enzymes, α-amylase and cellobiase, and then confirmed its utility with two recombinant proteins, amylosucrase and maltogenic amylase, expressed in E. coli. The Δ(fluorescence intensity) in DSF analysis accurately correlated with the concentration of the purified enzymes as well as the recombinant proteins in E. coli cell lysates. The main advantage of this method over other techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and green fluorescence protein (GFP) fusion proteins is that intact recombinant protein can be quantified without the requirement of additional chemicals or modifications of the recombinant protein. This DSF assay can be performed using widely available equipment such as a real-time polymerase chain reaction (RT–PCR) instrument, microplates or microtubes, and fluorescent dye. This simple but powerful method can be easily applied in a wide range of research areas that require quantification of expressed recombinant proteins.     

Analysis of nucleotide sugars from cell lysates by ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography

Analysis of nucleotide sugar metabolism is essential in studying glycosylation in cells. Here we describe practical methods for both extraction of nucleotide sugars from cell lysates and for their analytical separation. Solid-phase extraction cartridges containing graphitized carbon can be used for the purification of nucleotide sugars by using triethylammonium acetate buffer as a ion-pairing reagent for decreasing retention. After that they are separated by high-performance liquid chromatography using a C18 reversed-phase column and the same ion-pairing reagent for increasing retention. These new sample preparation and analysis methods enable good separation of structurally similar sugar nucleotides, compatibility with rapid evaporative concentration, and possibility to automation. Monitoring the production of GDP-deoxyhexoses in genetically engineered yeast and native bacterial cells are described here as specific applications.     

Identification of CDK2 substrates in human cell lysates

Background  

Protein phosphorylation regulates a multitude of biological processes. However, the large number of protein kinases and their substrates generates an enormously complex phosphoproteome. The cyclin-dependent kinases - the CDKs - comprise a class of enzymes that regulate cell cycle progression and play important roles in tumorigenesis. However, despite intense study, only a limited number of mammalian CDK substrates are known. A comprehensive understanding of CDK function requires the identification of their substrate network.     

Generation of superoxide ion in human red blood cell lysates

The generation of superoxide ion in human red blood cell lysates was investigated by an experimental method employing Cu,Zn superoxide dismutase as O2- scavenger and EPR to probe the oxidation state of the enzyme. The average value of the O2- flux in the erythrocytes of 8 normal individuals was (2.02 +/- 0.97) X 10(-8) M S-1. A progressive saturation of the rate of O2- production was found increasing PO2, KM = 1.04 X 10(-4) M, while the autoxidation of oxyhemoglobin did not contribute significantly to the measured O2- production.     

Two-dimensional peptide mapping of cross-linked type IX collagen in human cartilage

Type IX collagen is a quantitatively minor component of hyaline cartilage that is essential for the normal structural integrity of the tissue. Purification and analysis are difficult because the mature protein is insoluble as a cross-linked integral component of the fibrillar matrix. In order to view a peptide map of the total pool of type IX collagen in a cartilage sample, a selective method based on Western blot analysis was developed for displaying collagen IX peptides in a cyanogen bromide digest of tissue. Digests were partially resolved by reverse-phase HPLC, individual fractions were run on SDS-PAGE and then transblotted to membrane, and the collagen IX fragments were revealed using an anti-collagen IX rabbit antiserum. All major CB-peptides from alpha1(IX), alpha2(IX), and alpha3(IX) chains in the resulting two-dimensional display were identified by amino-terminal sequence analysis. Cross-linked peptides originating from sites of covalent interaction between collagen types IX and II and between IX and IX were also defined. By comparison with an analysis of soluble type IX collagen from chondrocyte culture medium, the results showed that the pool of type IX collagen molecules in fetal and adult human cartilage is extensively cross-linked intermolecularly at sites previously revealed by other methods using purified protein. This sensitive, direct method has the potential to screen for abnormalities in the content and properties of type IX collagen in tissue samples, for example, in the study of heritable chondrodysplasia syndromes and the pathogenesis of cartilage destruction in osteoarthritis.     

Two-dimensional electrophoresis of peptides from human-CHO cell hybrids containing human chromosome 21

Peptide expression influenced by human chromosome 21 was examined by comparing two-dimensional electrophoretograms of a human-hamster hybrid cell containing human chromosome 21 with its parent hamster cell and a revertant of the hybrid which had segregated the chromosome 21 genes for SOD-1, GARS, and a cytotoxic cell-surface antigen. Certain peptides were found in the hybrid but not in the hamster cell. Some, but not all, of these peptides segregated with markers for chromosome 21. Hamster peptides were also found which apparently were suppressed in the hybrid. Finally, one peptide was identified which was unique to the revertant cell. These findings may be of potential relevance to Down's syndrome.     

Two-dimensional electrophoresis database of fluorescence-labeled proteins of colon cancer cells

We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.     

Activation and inhibition of human cancer cell hyaluronidase by proteins

Results regarding hyaluronidase activity in tumor extracts or cell lines are subject to variations according to the method used for the assay and, sometimes, within an assay. Hyaluronidase was assayed at pH 3.8 in the culture medium of the human cancer-derived cell lines SA87 and H460M by several techniques: HPLC, Reissig technique, ELSA technique, and zymographic analysis. The optimal pH was between 3.3 and 4 in solutions at constant 150 mM sodium concentration. The enzyme was reversibly inhibited by sodium concentration over 200 mM. The activity of purified hyaluronidase increased in the presence of low concentrations of the specific HA-binding glycoprotein hyaluronectin, or of bovine serum albumin or immunoglobulins, or of human albumin, transferrin, or hemoglobin, showing that proteins cooperate in enzyme activity. The ELSA technique showed that optimal pH was slightly lower in the presence of HN than that with BSA. The optimal BSA concentration was determined with the ELSA technique at 0.1 g/liter, and excess of either protein inhibited hyaluronidase. When measured with the Reissig technique, the activity of purified enzyme in the presence of 0.1 g/liter BSA was up to fourfold that without BSA. The cooperative effect of BSA was visualized by zymography. We conclude that the total protein content of hyaluronidase solutions must be considered to correctly interpret quantitation of the enzyme in sera or tissue extracts because protein concentrations above 200 microg/liter lead to underestimation of the enzyme.     

Two-dimensional liquid chromatography study of the human whole saliva proteome

The human whole saliva proteome was investigated using two-dimensional liquid chromatography (2-DLC). The 2-DLC study was able to identify, with high confidence, 102 proteins including most known salivary proteins (35), and a large number of common serum proteins (67). Peptides from proline-rich proteins, abundant in saliva, had unusual cleavage sites and were frequently only partially tryptic. Three proteins not previously observed in human saliva were also detected. Significantly greater numbers of identified proteins, including high molecular weight, low molecular weight, and proline-rich proteins, were found with 2-DLC compared to previously reported two-dimensional gel electrophoresis studies.     

Two-dimensional gel analysis of proteins in cell lines from the central nervous system of larvalDrosophila

Summary High resolution two-dimensional gel electrophoresis was used to quantitatively analyze the patterns of protein synthesis in three different clones of a nerve cell line (ML-DmBG2) ofDrosophila melanogaster. When patterns of pulse-labeled proteins of the three different clones were compared, I observed quantitative variations affecting the rate of synthesis by twofold or more in 25–30% of the polypeptides and qualitative differences, always affecting less than 2% of the polypeptides. Patterns of protein synthesis were analyzed during the 24 d of culture, revealing both quantitative (increase or decrease; 40%) and qualitative (presence or absence; 3%) differences. More than 70 proteins synthesized in these cultures were secreted into the medium. Among them were two major groups of acidic proteins which disappeared with culture time. When cell lines and intact central nervous systems were compared, large differences in protein synthesis were observed. In fact, only 20% of the synthesized proteins were common to both isolated cells grownin vitro and the original nervous systemin vivo.     

Two-dimensional gel analysis of proteins from human trisomy 21 fetal liver tissue after DEAE-Sepharose chromatography

Summary Isoelectrofocusing two-dimensional polyacrylamide gel electrophoresis (IEF-2D-PAGE) offers the opportunity to detect typical alterations in the protein pattern from directly prepared liver tissue of fetuses with trisomy 21 and normal controls. The fractionation of the cell lysate by differential centrifugation into various subcellular components (nuclei, membranes, polyribosomes, cytoplasmic proteins) and fractionation of the proteins through DEAE-Sepharose chromatography allows detection of protein differences. In the 19th week of pregnancy it is possible to establish only three differences in the protein patterns between liver tissue from trisomy 21-fetuses and normal controls. All three proteins are synthesized in euploid controls at a higher level than in trisomy 21-liver tissue and are supposed to be consequences of primary gene dosage effects. The molecular mass of the individual proteins ranges from 14 kdaltons to 31 kdaltons. The data reported here raise the question whether some of the differences found by others represent different gene expression of cells under tissue culture conditions and/or of cells derived from different tissues, or developmental stages.     

Peptide mapping of proteins from acrylamide gels

A procedure is described by which proteins may be eluted from detergent gels, made radioactive by reaction with [¹²⁵I]sodium iodide, and analyzed by peptide mapping. The method permits a rapid and reliable means of identification of less than 10 μg of protein from a stained band.     

Peptide mapping of contractile proteins: Two-dimensional analysis of cyanogen bromide fragments on polyacrylamide gels

Peptide mapping of contractile proteins is necessary for correlation of the structual properties of these molecules with their distinct roles in various cell types. The large size of several of these polypeptides requires their cleavage by cyanogen bromide and the separation of the resulting products on a two-dimensional polyacrylamide-gel system to ensure reasonably complete peptide maps. Such a peptide map of rabbit skeletal muscle actin is in agreement with predictions from the amino acid sequence. The peptide map of rabbit skeletal muscle myosin can be resolved into maps of heavy and light meromyosins resulting from limited tryptic digestion of the myosin. Unique regions of the myosin map are occupied by one or the other of these segments. Analysis of native 105,000 M_r elam adductor paramyosin and its 94,000 M_r proteolytic products indicates that specific changes in peptide composition have occurred.     

Combinations of SPR and MS for characterization of native and recombinant proteins in cell lysates

Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni²⁺ for SPR analysis of the specific Ni²⁺ binding response and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein and of cauliflower were investigated for specific binding to the phosphopeptide ligands immobilized on a sensorchip by SPR. Subsequently, the bound protein was eluted and analyzed by MS for characterization of intact mass and peptide mass mapping.     

Classification of cancer cell lines using an automated two-dimensional liquid mapping method with hierarchical clustering techniques

A two-dimensional liquid mapping method was used to map the protein expression of eight ovarian serous carcinoma cell lines and three immortalized ovarian surface epithelial cell lines. Maps were produced using pI as the separation parameter in the first dimension and hydrophobicity based upon reversed-phase HPLC separation in the second dimension. The method can be reproducibly used to produce protein expression maps over a pH range from 4.0 to 8.5. A dynamic programming method was used to correct for minor shifts in peaks during the HPLC gradient between sample runs. The resulting corrected maps can then be compared using hierarchical clustering to produce dendrograms indicating the relationship between different cell lines. It was found that several of the ovarian surface epithelial cell lines clustered together, whereas specific groups of serous carcinoma cell lines clustered with each other. Although there is limited information on the current biology of these cell lines, it was shown that the protein expression of certain cell lines is closely related to each other. Other cell lines, including one ovarian clear cell carcinoma cell line, two endometrioid carcinoma cell lines, and three breast epithelial cell lines, were also mapped for comparison to show that their protein profiles cluster differently than the serous samples and to study how they cluster relative to each other. In addition, comparisons can be made between proteins differentially expressed between cell lines that may serve as markers of ovarian serous carcinomas. The automation of the method allows reproducible comparison of many samples, and the use of differential analysis limits the number of proteins that might require further analysis by mass spectrometry techniques.