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PubMed2000

Identification of Protein Spots in Silver-stained Two-dimensional Gels by MALDI-TOF Mass Peptide Map Analysis.

Protein spots in silver-stained two dimensional gels were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including i) In-gel reduction, alkylation and enzymatic digestion ii) Extraction and desalting by using a pipette tip containing a small C18 micro-column (ZipTip(TM)) iii) Direct MALDI-TOF mass analysis and protein database searching. The results demonstrate that single silver-stained protein spots in a 2-DE-gel could be identified rapidly by this procedure and the use of the ZipTip(TM) pipette tip could increase evidently the sensitivity of the MALDI-TOF analysis. By using this the procedure, 10 protein spots in a silver-stained gel of 2-DE of crude venom of the spider S.Huwena were analyzed and identified.

Journal Article↗
PubMed1986

Microscale elucidation of an N-linked glycosylation site by comparative high-performance liquid chromatographic peptide mapping.

The identification of specific sites of post-translational modifications of polypeptides is an important component in understanding protein structure-function relationships. This report describes the application of specific enzymatic methods of N-linked oligosaccharide removal (endo H and endo F) to the rapid microscale identification of specific sites of N-linked glycosylation. Following deglycosylation the protein is subjected to proteolytic digestion and comparative high-performance liquid chromatographic peptide mapping with an unmodified protein digest. Peptides from which oligosaccharide(s) have been removed exhibit a measurable increase in retention time on reversed-phase high-performance liquid chromatography and can be clearly identified. Once identified, the peptide can be subjected to direct protein microsequence analysis to elucidate the specific site of glycosylation. As illustrated here, these methods are compatible with microscale protein purification by sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by electroelution and are applicable to the 100-pmol range.

Amino Acid Sequence↗
PubMed1982

Characterization of myosin heavy chain by cyanogen bromide peptide maps.

Procedures have been developed for the preparation of pure myosin heavy chain (h-myosin) by preparative gel electrophoresis, and for the characterization of h-myosin by cyanogen bromide peptide mapping. Major sources of error are the oxidation of methionine and the proteolytic splitting of the chain during purification. These errors have been eliminated. A peculiar feature is the doubling or quadrupling of a peptide of molecular weight 17 000. The results show structural differences between isomyosins derived from myonal types within the same animal, as well as interspecies differences.

Animals↗
PubMedDate not supplied

[Analysis of species differences of radioactively labeled ceruloplasmin and albumin by means of peptide maps].

A procedure is developed for microanalysis of proteins, namely of human and rat ceruloplasmins as well as of their proteolytic fragments. The procedure involved electrophoretic purification of the peptides studied, labelling with 125I, tryptic hydrolysis and separation of the labelled peptides by cellulose thin-layer chromatography. Characteristics of the methods are described. Identification of individual polypeptide chains is reported. Specific localization of the labelled peptides on radioautogramms of the peptide maps was characterized. Analysis of human and rat ceruloplasmins, their fragments as well as human and rat blood serum albumins is presented as an example.

Animals↗
PubMed1977

Peptide mapping of 125I-labelled influenza virus proteins. Matrix proteins as markers in recombination.

We have examined the matrix proteins of A/Okuda/57, A/Finland/4/74 and A/New Jersey/8/76 viruses and several recombinant strains by radioiodination of the purified polypeptides followed by tryptic peptide mapping. The method is rapid and requires only small amounts of material. Reproducible differences were detected between the matrix proteins of the above parents and allowed origin of the matrix proteins of the recombinant viruses to be determined. The possible use of matrix protein identity as a marker in recombination work is discussed.

Chromosome Mapping↗
PubMed1980

Distinction between mouse DNA polymerases alpha and beta by tryptic peptide mapping.

Results presented here and in a previous paper (Tanabe et al. (1979) Biochemistry 18, 3401--3406) indicate that mouse beta-polymerase is a single polypeptide with an apparent molecular weight of 40,000. This polypeptide has now been analyzed by tryptic peptide mapping. Comparison of the results with identical analysis of mouse alpha-polymerase reveals that the tryptic peptides derived from the two enzymes are different. These results indicate that beta-polymerase is neither a subunit of alpha-polymerase nor a proteolytic degradation product of alpha-polymerase.

Animals↗
PubMed2005

Silicone/graphite coating for on-target desalting and improved peptide mapping performance of matrix-assisted laser desorption/ionization-mass spectrometry targets in proteomic experiments.

In two-dimensional gel electrophoresis-based proteomic experiments matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting is often the technique of choice in identifying proteins. Here, we present a novel surface coating technique for MALDI-MS targets that improves manual and automatic sample analysis. A mixture of silicone and graphite is spread in the form of a thin layer over the target. Due to the hydrophobicity of the coating, aqueous solutions can be applied to relatively small spots very precisely using a robotic system. At least four times more liquid can be concentrated on the same area compared to uncoated steel targets. alpha-cyano-4-hydrocinnamic acid crystallizes in form of very small crystals evenly distributed over the surface. The search for "hot spots" during the analysis is not necessary, which supports the automatic acquisition of data. The homogeneous crystal layer can be very effectively washed on-target without encountering major sample losses. This efficient washing and the focused application of aqueous samples replace expensive and time-consuming reversed phase micro column based sample clean-ups. When analyzing peptide mixtures, the signal intensities are up to five times higher than with preparations of the same un-desalted samples on steel targets, since four times more sample can be loaded. The mass resolution remains unaffected by the surface coating. After usage the coating can be removed, followed by a new coating avoiding any carry-over of sample to the next analysis. All these properties make the precoating of MALDI-MS targets with a silicone/graphite layer an ideal technique for routine analysis in large-scale proteomic experiments.

Autoanalysis↗
PubMed1996

Comparison of antibodies raised against the peptide 10-24 of chicken riboflavin carrier protein (cRCP) by classical and multiple antigen peptide (MAP) approaches.

Riboflavin carrier protein is an essential protein required for the growth and development of the embryo and hence for the maintenance of pregnancy. Our efforts to delineate the antigenic determinants of chicken riboflavin carrier protein (cRCP) resulted in the identification of a bioneutralization epitope in the region 10-24 of cRCP. The present work compares the properties of the antibodies raised against the peptide epitope by classical and multiple antigen peptide (MAP) system approaches. The extent of cross-reaction of the antibodies to the MAP construct with the parent protein was found to be significantly less as compared to the antibodies raised against the peptide-diphtheria toxoid conjugate. Furthermore, the bioneutralizing ability of the antisera to the MAP construct was also found to be very poor. The results suggest that there are serious limitations in the ability of antibodies raised against MAP constructs to cross-react with the native proteins.

Amino Acid Sequence↗
PubMed1974

Peptide mapping of aminoacyl-tRNA synthetases: evidence for internal sequence homology in Escherichia coli leucyl-tRNA synthetase.

Most aminoacyl-tRNA synthetases contain polypeptide chains of about either 50,000 or 100,000 daltons. Peptide mapping of tryptic, chymotryptic, or Staphylococcus aureus acid protease digests of seryl-tRNA synthetase (100,000, dimer) and leucyl-tRNA synthetase (100,000, monomer) from E. coli was done after selective modification of lysine residues with [(14)C]succinic anhydride or of methionine residues with [(14)C]iodoacetate. By use of thin-layer electrophoresis and chromatography on silicagel or cellulose plates followed by radioautography it was possible, depending upon the specific activity of the reagent used, to detect radioactive peptides obtained from as little as l mug of protein.Seryl-tRNA synthetase gave the correct number of tryptic peptides expected for a dimer of identical subunits. Leucyl-tRNA synthetase, on the other hand, gave roughly half the number of radioactive tryptic, chymotryptic, and acid protease peptides expected from the lysine, arginine, and methionine content of the 100,000 monomer. We have interpreted these results as indicating that extensive internal homology exists among lysine- and methionine-containing peptides within the leucyl-tRNA synthetase. The simplest conclusion that can be drawn from these observations is that the NH(2)- and COOH-terminal halves of leucyl-tRNA synthetase and perhaps other synthetases of 100,000 molecular weight may have evolved through a process of gene duplication and fusion, followed by limited diversification by way of amino-acid substitutions accumulating during evolution.

Amino Acyl-tRNA Synthetases↗
PubMed1980

BALB/c myeloma retroviruses: peptide mapping and immunological analysis of the pp12 structural protein.

We have compared the pp12 structural protein of the MO-21 and FL-1 BALB/c myeloma retroviruses with the pp12 of several prototype retroviruses. Chymotryptic peptide maps of 125I-labeled, immune-precipitated pp12 proteins revealed that the MO-21 and FL-1 proteins can be distinguished from one another. The MO-21 pp12 most closely resembled the NIH-xenotrophic virus pp12, and the FL-1 pp12 most closely resembled the pp12 of BV-2 and WN 1802 B. Competition radioimmunoassay studies showed that the MO-21 and FL-1 pp12 proteins are also antigenically distinct from one another and that both contain pp12 antigenic determinants of a xenotropic virus. These data support our proposal that these two BALB/c viruses contain a gag gene that was generated by recombination between endogenous eco- and xenotropic viral sequences.

Animals↗
PubMed1995

Epitopic regions for antibodies against tumor necrosis factor alpha. Analysis by synthetic peptide mapping.

The location of biologically relevant epitopes on human tumor necrosis factor alpha (hTNF-alpha) was evaluated by testing the immunoreactivity of anti-TNF-alpha antibodies against 149 sequential, overlapping octamer peptides. A goat polyclonal antibody raised against recombinant hTNF-alpha, which neutralizes hTNF-alpha biological activities, reacted with oligopeptides corresponding to hTNF-alpha residues 7-11, 17-23, 30-39, 42-49, 106-112, and 135-142. A possible assembled epitopic region (residues 25, 27, and 144) for neutralizing murine monoclonal antibodies designated 11D7G4 and 9C4G5 was deduced from the fact that they bound to tripeptides, mimicking a discontinuous epitope. These antigenic regions wer found to include of adjoin poorly conserved amino acids and they were located in the turns between beta-sheets on the surface of the molecule. Three of the sequential epitopic regions and an assembled region were closely related to the receptor binding sites proposed in several other studies. These antibodies appear to neutralize TNF-alpha activities by directly masking receptor binding sites.

Animals↗
PubMed1986

High-performance liquid chromatographic peptide mapping and amino acid analysis in the sub-nanomole range.

By ensuring adequate gradient mixing and eliminating all major artifact peaks we have been able to obtain reproducible high-performance liquid chromatographic tryptic peptide maps on less than 50 pmol of protein. Likewise, a 10-20 fold improvement in the sensitivity of amino acid analysis has been achieved by analyzing the phenylthiocarbamyl derivatives of the free amino acids rather than the free amino acids themselves. This approach enables accurate amino acid compositions to be obtained on less than 50 ng of protein, providing that a simple correction is made for the background level of serine and glycine. We have been able to reduce the background level of these two amino acids to ca. 10 pmol each per sample by incinerating the hydrolysis tubes at 500 degrees C prior to introduction of the sample and by using gas-phase as opposed to liquid-phase hydrolysis. Background corrections are not necessary when over 500 ng of protein are hydrolyzed. With this amount of protein, amino acid compositions, based on phenylthiocarbamyl amino acid analyses were, on average, found to be accurate to within +/- 10%.

Amino Acids↗
PubMed1993

Peptide-mapping of three neutralizing epitopes into predicted biologically active sites of human interferon-alpha 2.

Immunologically less reactive but functionally relevant structures were identified on human interferon (IFN)-alpha 2 by three neutralizing monoclonal antibodies (mAb). The binding sites of these mAbs were mapped using a set of synthetic peptides that covered the amino acid sequence of two predicted biologically active segments in the regions 31-53 and 63-85 of IFN-alpha 2. We measured the capacity of fragments to inhibit the IFN-neutralizing activity of mAbs and located three linear epitopes around residues 42-53, 63-76 and 77-85 of the IFN-alpha 2 molecule.

Amino Acid Sequence↗
PubMed1985

One-dimensional peptide mapping of the subunits of pertussis toxin.

Pertussis toxin (pertussigen) purified from the cytoplasmic fraction of Bordetella pertussis strain 18334, phase 1, consisted of five subunits which included an additional subunit (S1a) not previously reported. Subunits S1, S1a and S2 showed extensive structural homology when analysed by one-dimensional peptide mapping, indicating that the latter two were probably derived from proteolytic cleavage of the largest subunit, S1. Subunits S3 and S4,5 generated only a limited number of peptides following chemical and enzymic degradation, but these subunits differed structurally from each other and from those showing structural homology.

Bacterial Toxins↗
PubMed1999

Effect of adjuvant composition on immune response to a multiple antigen peptide (MAP) containing a protective epitope from Neisseria meningitidis class 1 porin.

A variety of adjuvants with the potential for use with experimental human vaccines were used for immunisation of mice, in an attempt to augment the humoral immune response to a multiple antigen peptide (MAP) containing a protective epitope from the sero-subtype specific class 1 porin protein of Neisseria meningitidis, in tandem with a Th-cell epitope. Surface plasmon resonance showed that combinations of the immunomodulators pluronic block co-polymer, muramyl dipeptide and monophosphoryl lipid A (MPL), increased the magnitude and avidity of the immune response in comparison with both Al(OH)3 and Freund-type adjuvants. In addition, the incorporation of MPL was essential for the induction of a broad distribution of antibody isotypes. The antibodies induced recognised the native protein in meningococcal outer membranes in a subtype-specific manner. The formulations containing these multiple immunomodulators which have already been used in human phase I/II trials with experimental vaccines, are candidates for inclusion in future human vaccines based on synthetic peptides containing defined, protective epitopes.

Adjuvants, Immunologic↗
PubMed1977

Peptide mapping of the lambda-like chains of the BASILEA rabbits.

The recently established mutant strain of rabbits, named BASILEA, lacks the k polypeptide L-chain which is replaced by a lambda-like type (bas). This is evident from the amino acid composition and peptide maps of the bas L-chain when compared to the L-chains of the rabbits suppressed for the expression of the b locus allotypes.

Amino Acids↗
PubMed1990

Fine analysis of intrathecally synthesized anti-HIV IgG1-4 by peptide mapping.

ELISAs utilizing HIV-derived synthetic peptides as antigen were used to analyze serum and CSF IgG1-4 from 15 HIV infected patients. Intrathecally synthesized IgG1-4 reactive to one or several HIV-derived peptides were detectable in 12 of 15 patients. Intrathecally synthesized anti-peptide IgG was more common in patients with neurological symptoms than in those without. CSF reactivity not paralleled by serum reactivity was detected to HIV-peptides in 4 patients. IgG1-4 to gp41 was relatively more frequent in the CNS than IgG1-4 to gag. Intrathecal IgG synthesis to the gp120 peptide was not detected in any patient. The anti-peptide responses were dominated by IgG1. Intrathecal IgG2 and 4 synthesis was found in 2 and 5 patients, respectively. IgG3 synthesis intrathecally was not detected in any of the patients. ELISAs detecting IgG1-4 to HIV-derived synthetic peptides are feasible to analyze the fine specificities of intrathecal IgG. The mapping of idiotypes and isotypes of IgG synthesized in the CNS will increase the possibilities of elucidating B-cell regulation in the CNS and which viral components evoke immune responses.

Gene Products, gag↗