Proteomic analysis of protein expression profiles during Caenorhabditis elegans development using two-dimensional difference gel electrophoresis

Coordinated protein expression is critical for the normal execution of animal development. To obtain overall proteome profiles during animal development, a small free-living soil nematode, Caenorhabditis elegans, was used as a model and the developmental changes of protein expressions were analyzed using two-dimensional difference gel electrophoresis. Protein samples from six developmental stages were prelabeled with fluorescent cyanine dyes and separated on two-dimensional electrophoresis gels. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the developmental expression profiles of 231 spots representing 165 proteins. About a quarter of the identified proteins were expressed in multiple spots with different isoelectric points, suggesting a certain proportion of proteins were variously modified. This notion was supported by the observation that about a third of the multispot proteins were stained positive for a phosphoprotein specific dye. While a fairly large number of the proteins showed little alteration in their expression profiles during development, about 40 proteins were found to be significantly either up- or down-regulated between the embryos and newly hatched L1 larvae. Down-regulated proteins included those related to the cell cycle such as MCM-7, PCN-1, and the mitotic checkpoint protein, while up-regulated proteins included structural proteins such as actins, LEV-11, DIM-1, VAB-21, metabolic enzymes such as ATP synthase, ALH-12, fluctose-1,6-bisphosphate aldolase and GPD-3, and galectins. A standard proteome map was obtained where the defects in the mutations of developmental genes and the effects of reagents on the development in C. elegans were analyzed.     

Isolation ofSpiroplasma citri membranes and characterization of membrane proteins by two-dimensional gel electrophoresis

This paper describes a method for isolating plasma membranes fromSpiroplasma citri and for comparing membrane and cytoplasmic proteins by two-dimensional gel electrophoresis. Plasma membranes ofS. citri were stabilized against fragmentation by coating cell with Concanavalin A just prior to lysis. After lysis of the cells by ultrasonic irradiation, membranes were purified by differential centrifugation and step gradients. The purified fraction, which consisted essentially of extended sheets of membranes, exhibited membrane-boundpara-nitrophenylphosphatase specific activity 1.5-fold over that of the whole-cell lysate. Only traces of soluble NADH oxidase were present in the membrane preparation. The latter fraction appeared homogeneous upon sorbitol density gradient centrifugation and banded at an equilibrium density of 1.107 g/ml. The plasma membrane proteins were then analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 40 different proteins were detected in the membrane preparations. By comparison with the patterns obtained for whole-cell extracts and cytoplasmic fractions, a protein map ofS. citri could be established in which membrane and cytoplasmic proteins were identified.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Background  

Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response.     

Induction of leucine aminopeptidase by interferon-gamma. Identification by protein microsequencing after purification by preparative two-dimensional gel electrophoresis.

The protein previously called "Mr approximately 50/pI approximately 6.9," which we observed to be induced by the immunoregulatory cytokine interferon (IFN)-gamma in human fibroblasts, was purified from a total cell lysate by preparative two-dimensional gel electrophoresis and identified by partial amino-terminal sequencing as leucine aminopeptidase (LAP), a 53-kDa cytosolic exopeptidase. Induction of LAP protein by IFN-gamma, confirmed by immunoblotting with an antiserum raised against bovine lens LAP, is a consequence of induction of LAP mRNA and occurs in all four human cell lines examined: HS153 fibroblasts, ACHN renal carcinoma, A549 lung carcinoma, and A375 melanoma. Induction of LAP mRNA is a secondary response to IFN-gamma, blocked by inhibition of protein synthesis with cycloheximide.     

Isolation, characterization and immunolocalization of phosphorylcholine-substituted glycolipids in developmental stages of Caenorhabditis elegans.

Caenorhabditis elegans displays three neutral glycosphingolipids with structural homology to glycosphingolipids from the porcine nematode parasite, Ascaris suum. The present findings extend the degree of structural conservation between the two nematode species to glycosphingolipids with a phosphodiester substitution. Using a combination of hydrofluoric acid pretreatment, immunochemical characterization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, three zwitterionic, phosphorylcholine-substituted glycosphingolipids could be identified in the neutral glycolipid fraction of C. elegans. The components were isolated as their zwitterionic, phosphorylcholine-substituted, pyridylaminated oligosaccharides by HPLC. Structural analysis was performed using hydrofluoric acid treatment, partial acid hydrolysis, methylation analysis, gas chromatography-mass spectrometry, cleavage with exoglycosidases and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures are proposed as: component Nz1, GalNAc(beta1-4)[phosphorylcholine]GlcNAc(beta1-3)Man(beta1-4)Glc-cera mide; component Nz2, Gal(alpha1-3)GalNAc(beta1-4)[phosphorylcholine]-GlcNAc(beta1-3)Man(be ta1-4)Glc-ceramide; and component Nz3, Gal(beta1-3)- Gal(alpha1-3)GalNAc(beta1-4)[phosphorylcholine]GlcNAc(beta1-3)Man(bet a1-4)Glc-ceramide. The oligosaccharide core is characteristic of the biosynthetic arthro-carbohydrate series of protostomial glycosphingolipids. The ceramide moiety was specified by a d17 : 1 sphingoid-base with iso-branching and anteiso-branching, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids. Analysis of the spatial and temporal expression of the phosphorylcholine epitope, during embryonic and postembryonic development, showed it to be localized predominantly in seam cells and basement membranes, respectively. In early embryonic ontogenesis the phosphorylcholine epitope was only lipid bound, while in late embryonic and postembryonic development this epitope was both lipid bound and protein bound.     

Approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography,gel electrophoresis and mass spectrometry

A combination of separation and identification techniques was used to rapidly and reproducibly analyze influenza vaccine constituents. Size-exclusion HPLC analysis reduced significantly the complexity by providing a constituents profile according to size. Significantly, no sample treatment was required prior to analysis thus eliminating a potential source of artifacts and degradation. Distinct profiles were associated with influenza strains as well as with vaccines from different manufacturers. Samples analyzed over several years allowed evaluation of method performance and provided stability-indicating data relating to the structural integrity of separated components. Collected chromatographic peaks were identified by gel electrophoresis and MALDI/MS of tryptic digests from excised gel bands. The challenge in obtaining high quality analytical data from complex mixtures clearly demonstrated the value of separation steps prior to MS identification. The method presented here is not intended to replace existing methodology; it is intended to provide a product specific profile to be used as a rapid screen for manufacturer, year (for annual influenza vaccines), stability or counterfeit product. It is a new screening method that provides a rapid and robust indication of products which require further investigation as a result of a deviation in their characteristic profile. Until now this tool did not exist.     

Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans

Dauer formation in Caenorhabditis elegans is regulated by at least three signaling pathways, including an insulin receptor-signaling pathway. These pathways were defined by mutants that form dauers constitutively (Daf-c) at 25 degrees. Screens for Daf-c mutants at 25 degrees have probably been saturated, but failed to identify all the components involved in regulating dauer formation. Here we screen for Daf-c mutants at 27 degrees, a more strongly dauer-inducing condition. Mutations identified include novel classes of alleles for three known genes and alleles defining at least seven new genes, hid-1-hid-7. Many of the genes appear to act in the insulin branch of the dauer pathway, including pdk-1, akt-1, aex-6, and hid-1. We also molecularly identify hid-1 and show that it encodes a novel highly conserved putative transmembrane protein expressed in neurons.     

Affinophoresis in two-dimensional agarose gel electrophoresis: specific separation of biomolecules by a moving affinity ligand

Affinophoresis is an electrophoretic separation technique for biomolecules which uses an affinophore. An affinophore is a macromolecular polyelectrolyte bearing affinity ligands. It migrates rapidly in an electric field, and consequently the electrophoretic mobility of molecules having affinity for the ligand is specifically changed. This technique has now been incorporated in two-dimensional agarose gel electrophoresis in a procedure which utilizes normal electrophoresis in the first dimension and affinophoresis in the second dimension. Proteins which do not have affinity for the ligand migrate to locations along a diagonal line passing through the origin, whereas proteins which have affinity are carried away from the line by the affinophore. Accordingly, molecules having affinity for the ligand can be readily assigned. Trypsins contained in Pronase and pancreatin were separated by this procedure using an affinophore bearing a competitive inhibitor for trypsin, benzamidine, on a polyanionic molecule (a polyacrylic acid derivative).     

Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping

Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3–10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight > 10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots.     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis

Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834–840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746–2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological activities to fully understand the mechanisms of Cu tolerance and toxicity in plants.     

Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.     

Identification of surface proteins of Helicobacter pylori by selective biotinylation,affinity purification,and two-dimensional gel electrophoresis

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.