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PubMed1999

Use of vapor-phase acid hydrolysis for mass spectrometric peptide mapping and protein identification.

A method for mass spectrometric peptide mapping was developed, based on hydrolysis of a solid protein by acid vapor followed by mass spectrometric analysis of the cleavage products. The method is applicable to lyophilized samples as well as proteins present in gels after separation by SDS-PAGE. The cleavage specificity was established using a number of standard proteins. Three different types of cleavages were observed: specific internal backbone cleavages at Asp, Ser, Thr, and Gly and N- and C-terminal sequence ladders. On the basis of the observed cleavage characteristics, a strategy for protein identification based on the peptide mass maps was developed. The identification strategy utilizes the specific internal backbone cleavages as well as the partial sequence information, obtained from the sequence ladders.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular characterization of a conformational epitope of hen egg white lysozyme by differential chemical modification of immune complexes and mass spectrometric peptide mapping.

A new approach for the characterization of conformationally dependent epitope structures in protein antigens is described using differential chemical modification of immune complexes in combination with mass spectrometric peptide mapping analysis. Well-established methods for epitope characterization are frequently not applicable to conformationally dependent epitopes, and direct methods of structure analysis such as X-ray crystallography of immune complexes have been successful only in a few cases. Our approach combines tertiary structure-selective chemical modification of immune complexes with the molecular characterization of reaction products by mass spectrometric peptide mapping. The comparison of the modification pattern of free and antibody-bound antigen provides the identification of residues protected from modification by the antibody. These residues hence are characterized as part of the epitope structure. The well-characterized hen egg white lysozyme and a corresponding monoclonal IgM-type antibody were investigated as a model system. Specific modification reactions for arginine, lysine, and tyrosine residues were performed, and the modification sites in free and antibody-bound antigen were determined by mass spectrometric peptide mapping. The R14 residue and residues K13 and K96 in the antibody-bound lysozyme were found to be protected from modification, comprising a surface of spatially adjacent residues by folding of the native protein. In contrast, other K and R residues as well as Y20 and Y23 showed no significant shielding from modification in the immune complex. These results provided an estimation of the molecular epitope surface area of native lysozyme.

Animals↗
PubMedDate not supplied

Peptide mapping of mammalian brain protein h3 in subsets of tissues and ligand binding studies.

In this report the structure of the novel polypeptide h3, isolated from subsets of tissues of a single species, and the structural similarity between interspecies h3 were investigated by peptide mapping of enzymatic and chemical cleavage fragments of h3 in one-dimensional SDS-PAGE; the peptide maps were commented on in comparison with the known sequence of 21 kDa protein, a h3-like ox brain protein. The following results were obtained: peptides generated by chymotrypsin, protease XX, BNPS-skatole and CNBr cleavage of different tissues in a single species were strikingly identical, whereas peptide maps obtained from analogue tissues in different species revealed slight structural differences. Possible ligand-h3 binding was studied by comparing the c.d. spectra of native h3, and h3 incubated with several phospholipids. Given the presence of h3 or h3-like protein in rat and human platelets, h3 was also assessed in platelet aggregation in the presence of h3 and specific anti-h3 antiserum. So far, the results emphasize the unique intra- and interspecies molecular form of h3, allow us to assign to known amino acid sequence of 21 kDa to a large extent to human h3, but do not identify h3 as a phospholipid binding protein.

Androgen-Binding Protein↗
PubMed1990

Sensitive blotting assay for the detection of glycopeptides in peptide maps.

A dot blotting assay using digoxigenin hydrazide (Glycan detection kit, Boehringer Mannheim Biochemicals) was used to screen an endoproteinase Lys-C peptide map of ribonuclease B for the presence of glycopeptides. The carbohydrate content of the identified glycopeptide fraction was then further characterized by monosaccharide analysis using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). The tandem use of a hydrazide dot blotting technique to screen peptide maps for glycopeptides and subsequent use of HPAE-PAD to identify the monosaccharide composition of glycopeptide hydrolyzates proved to be a quick, sensitive and reliable method for identifying glycopeptides and analyzing their glycan composition without derivatization of the carbohydrate.

Animals↗
PubMed1986

Identification of the C-terminal portion of a protein by comparative peptide mapping.

A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.

14-3-3 Proteins↗
PubMed1984

Myosin light chains of avian and mammalian slow muscles: peptide mapping of 2S light chains.

The 2S light chains of mammalian and avian slow muscle myosin, indistinguishable by two-dimensional gel electrophoresis, have been examined by peptide mapping. The fragments obtained with S. aureus V8 protease were analysed either by gel electrophoresis or by reverse-phase high performance liquid chromatography. The peptide maps of avian 2S light chains contain fragments distinct from those of mammalian 2S light chains. Chicken and turkey LC2S appear to be more similar to each other than those from mammalian species (rat and rabbit). These results are in agreement with the relative phylogenetic distances among the four species studied here. The 2S light chain of slow muscle represent further examples of polypeptides which comigrate in two-dimensional gel electrophoresis in spite of their different peptide maps.

Animals↗
PubMed1982

Molecular epidemiology of tick-borne encephalitis virus: peptide mapping of large non-structural proteins of European isolates and comparison with other flaviviruses.

Nine virus-specified proteins were identified by SDS-polyacrylamide gel electrophoresis in [35S]methionine-labelled chick embryo cells infected with tick-borne encephalitis (TBE) virus by comparison with mock-infected cells. These proteins were designated P91, p74, p72, P67, GP53(E), P47, p25, P15(C) and P14.5 according to their molecular weights. Peptide mapping of P91, P67, GP53(E) and P47 from TBE virus-infected cells, as well as those of the corresponding proteins from West Nile virus (WNV)-infected cells (previously termed NV5, NV4, V3 and NV3), demonstrated the uniqueness of these proteins. Almost no subtype variability, with respect to the pattern of intracellular proteins, was found when isolates of TBE virus from Austria, Switzerland, Germany, Finland and Czechoslovakia were compared. Peptide mapping of NV5 (P91) and NV4 (P67) from all these isolates using limited proteolysis with alpha-chymotrypsin and V8 protease revealed completely identical patterns, thus extending our observations that TBE virus seems to represent a very stable member of the flavivirus genus, which was based on the lack of variation found with the structural glycoprotein. On the other hand, the Far Eastern subtype of TBE virus and the closely related louping-ill virus could not only be differentiated from the Western subtype by differences in the peptide maps of their structural glycoprotein but also in those of the non-structural protein NV5, i.e. subtype or subgroup variations are not confined to the virion surface glycoprotein. WNV and Murray Valley encephalitis virus (MVEV) revealed the expected heterogeneity of virus-specified proteins found in cells infected with different flaviviruses. It is especially interesting that also the largest non-structural protein, NV5, is subject to this heterogeneity, ranging in mol. wt. from 91 000 for TBE virus to 98 000 for MVEV and that also the peptide maps of NV5, as well as those of NV4, were unrelated. These proteins, therefore, revealed a variability between serologically distinct flaviviruses similar to that observed with the structural glycoprotein.

Encephalitis Viruses, Tick-Borne↗
PubMed2004

Database search post-processing by neural network: Advanced facilities for identification of components in protein mixtures using mass spectrometric peptide mapping.

Database search post-processing by neural network was employed in peptide mapping experiments. The database search was performed using both the known algorithms and score functions, such as Bayesian, MOWSE, Z-score, correlations between calculated and actual peptide length fractional abundance, and, in addition, the probability of protein digest pattern in peptide fingerprint, all embedded in locally developed program. The new signal-processing algorithm based on neural network improves signal-noise separation and is acceptable for automatic protein identification in mixtures. Its power was tested on Helicobacter pylori protein inventory after preceding protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increase in protein identification success rate was observed, and about 100 proteins were identified with no need of human participation in database search estimation.

Algorithms↗
PubMed2001

Peptide mapping by capillary zone electrophoresis: how close is theoretical simulation to experimental determination.

A multi-variable computer model is presented for the prediction of the electrophoretic mobilities of peptides at pH 2.5 from known physico-chemical constants of their amino acid residues. The model is empirical and does not claim any theoretical dependencies; however, the results suggest that, at least at this pH, peptides may be theoretically represented as classical polymers of freely joined amino acid residues of unequal sizes. The model assumes that the electrophoretic mobility can be represented by a product of three functions that return the contributions of peptide charge, length and width, respectively to the mobility. The model relies on accurate experimental determination of the electrophoretic mobilities of a diverse set of peptides, by capillary zone electrophoresis (CZE), at 22 degrees C, with a 50 mM phosphate buffer, at pH 2.5. The electrophoretic mobilities of a basis set of 102 peptides that varied in charge from 0.65 to 16 and in size from two to 42 amino acid residues were accurately measured at these fixed experimental conditions using a stable 10% linear polyacrylamide-coated column. Data from this basis set was used to derive the peptide charge, length, and width functions respectively. The main purpose of this endeavor is to use the model for the prediction of peptide mobilities at pH 2.5, and for simulation of CZE peptide maps of protein digests. Excellent agreement was obtained between predicted and experimental electrophoretic mobilities for all categories of peptides, including the highly charged and the hydrophobic. To illustrate the utility of this model in protein studies it was used to simulate theoretical peptide maps of the digests of glucagon and horse cytochrome c. The resulting maps were compared and contrasted with their experimental counterparts. The potential of this approach and its limitations are discussed.

Amino Acid Sequence↗
PubMed1986

Structural differences of microtubule associated proteins from brain probed by tryptic peptide mapping.

Microtubules were purified from porcine brain by two cycles of temperature-dependent assembly and disassembly, then microtubule associated proteins, MAP-1, MAP-2, and tau, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional tryptic peptide maps of radioiodinated polypeptides were compared with each other by means of mixed sample experiments, and the following results were obtained. Subspecies of MAP-1 (355-345 and 325 kDa) showed about 33% homology in the tryptic peptide maps. Structural homology of MAP-1 and MAP-2 was very low; only 3 out of 40 peptide spots of MAP-2 were identical with those of MAP-1-C. Subspecies of tau proteins (65 and 60 kDa) were very closely related. Structural similarity between MAP-2 and tau was very low. MAP-1 from porcine brain and rat brain showed very high structural homology.

Animals↗
PubMed1989

Peptide mapping by CNBr fragmentation using a sodium dodecyl sulfate-polyacrylamide minigel system.

The method of peptide mapping by sodium dodecyl sulfate-gel electrophoresis following partial protein fragmentation with cyanogen bromide was adapted for a polyacrylamide minigel system. The combined use of the discontinuous gel electrophoresis system of J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271) and a vertical polyacrylamide minigel system produced the following advantages over other procedures: (a) the ability to resolve cyanogen bromide cleavage fragments over a broad molecular mass range while yielding very sharp protein staining bands; (b) well-defined peptide maps are produced with as little as 2 micrograms of protein; (c) less time is required to perform fragmentation with cyanogen bromide, to equilibrate the gel slices in sodium dodecyl sulfate buffer, as well as to perform the electrophoresis; and (d) the cyanogen bromide fragmentation patterns are highly reproducible.

Cyanogen Bromide↗
PubMed1984

Two-dimensional peptide mapping by polyacrylamide-gel electrophoresis with limited proteolysis in SDS.

The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility.

Animals↗
PubMed1985

Peptide mapping of bovine pancreatic ribonuclease A by reverse-phase high-performance liquid chromatography. II. A two-dimensional technique for determination of disulfide pairings using a continuous-flow disulfide-detection system.

A procedure, developed for the cleavage and reversible blocking of disulfide bonds of proteins by S-sulfonation in preparation for peptide mapping, was applied to ribonuclease A. The complete peptide maps of sulforibonuclease A using limited Staphylococcus aureus protease digestion, tryptic digestion, and tryptic followed by chymotryptic digestion are presented. A description is given of an adaptation of the sulfonation procedure which forms the basis of a sensitive (5-pmol detection limit) and quantitative (+/- 5%) disulfide-detection system for the continuous monitoring of HPLC column effluents for disulfide-containing compounds. The sulfonation procedure, peptide maps, and disulfide-detection system are the key ingredients in a two-dimensional reverse-phase HPLC technique for the determination of disulfide pairings. The applicability of this technique is demonstrated by determining the known disulfide pairings of ribonuclease A. It is also shown that there is no disulfide interchange under the digestion conditions used. This technique is suitable for determining the distributions of disulfide pairings in the intermediates present in the oxidative folding of disulfide-containing proteins.

Animals↗
PubMed1991

Recognition by peptide mapping of three different structural groups of outer membrane protein YOP-1 of Yersinia enterocolitica and Yersinia pseudotuberculosis.

The structural relation of YOP-1 of "european" and "american" Yersinia enterocolitica serotypes O:3, O:9, O:5,27, and O:8 and O:20, respectively, and Y. pseudotuberculosis serotypes I, II, and III was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping using Staphylococcus aureus protease V8. Apparent molecular weights of YOP-1 ranged from 206,000 (O:3) to approx. 180,000 (O:8). According to their respective peptide maps YOP-1 of the "european" and "american" Y. enterocolitica serotypes and Y. pseudotuberculosis serotypes could be assigned to three different groups. Evaluation of several isolates of Y. enterocolitica serotypes O:3, O:9, and O:8 by peptide mapping indicated that YOP-1 is conserved within a serotype. However, one serotype O:8 isolate differed from the consensus peptide pattern of the other serotype O:8 and O:20 isolates. The similarity of the peptide patterns of Yersinia serotypes which predominate in certain geographical locations, i.e., "european" and "american" Y. enterocolitica serotypes, suggest common evolution of YOP-1 of these serotypes independent of the evolution of the other serotypes.

Adhesins, Bacterial↗
PubMed1977

Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin.

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.

Arboviruses↗
PubMed1985

Peptide mapping of envelope-related glycoproteins specified by the flaviviruses Kunjin and West Nile.

Glycoproteins detected in Vero cells infected by the flaviviruses West Nile and Kunjin were examined by gel electrophoresis and peptide mapping. Two major glycoproteins, gp66 and gp54, were observed in West Nile virus-infected cells labelled for short time periods with [3H]mannose. A third glycoprotein, gp58, was present in smaller amounts. Pulse-labelling experiments suggested that gp66 was a precursor of gp54. Peptide mapping of [3H]leucine-labelled gp66, gp54 and the envelope glycoprotein E of West Nile virus demonstrated that gp66 and gp54 were related to E, and that the peptides of gp54 were a subset of those of gp66. Peptide mapping of the corresponding Kunjin virus-specified glycoproteins (gp66, gp59 and gp53) showed that the [3H]leucine-labelled peptides of gp53 and gp59 were similar and were contained within gp66. Since we have shown previously that gp59 and gp53 are related to E of Kunjin virus, we conclude that cells infected by West Nile or Kunjin viruses contain a similar set of E-related glycoproteins.

Flavivirus↗
PubMed2003

Peptide mapping by reversed-phase high-performance liquid chromatography employing silica rod monoliths.

In this paper, a general procedure is described for the generation of peptide maps of proteins with monolithic silica-based columns. The peptide fragments were obtained by tryptic digestion of various cytochrome c species with purification of the tryptic fragments achieved by reversed-phase high-performance liquid chromatographic methods. Peak assignment of the various peptides was based on evaluation of the biophysical properties of the individual peptides and via mass spectrometric identification. The performance of several different monolithic sorbents prepared as columns of identical cross-sectional dimensions were investigated as part of these peptide mapping studies and the data evaluated by applying solvent strength theory. These studies revealed curvilinear dependencies in the corresponding relative resolution maps. These findings directly impact on the selection of specific sorbent types or column configurations for peptide separations with silica rod monoliths. Moreover, the influence of variations in the amino acid sequence of the cytochrome cs were evaluated with respect to their effect on intrinsic hydrophobicity, the number of experimental observed tryptic cleavage sites, detection limits of the derived fragments in relation to their molecular size, and the chromatographic selectivity and resolution of the various peptides obtained following enzymatic fragmentation of the parent protein. Finally, the scope of these approaches in method development was examined in terms of robustness and efficiency.

Chromatography, High Pressure Liquid↗
PubMed1981

The use of 2-dimensional CNBr peptide maps for the analysis of crosslinked peptides in bone collagen.

CNBr peptides from insoluble bovine cortical bone collagen were analyzed using a 2-D mapping technique. The major type 1 collagen CNBr peptides were detected by fluorography after general-labelling with 3H-NaBH4 in dimethyl-formamide. These maps were similar to those visualized by coomassie blue staining and demonstrated a proportional decrease of alpha 1CB6. New groups of peptides, different from those normally present in soluble type I collagen were detected. Some of these peptides were slightly larger and more acidic than alpha 1CB6 and were highly labelled when the demineralized bone was specifically labelled for the presence of aldehydes and crosslinks with 3H-NaBH4 in a phosphate buffer, pH 7.4. Based on the size and charge characteristics of these specifically labelled peptides, they were tentatively identified as crosslinking peptides containing different combinations of alpha 1CB6, alpha 1CB0,1 and alpha 1CB5. The specificity of the labelling method using 3H-NaBH4 in phosphate buffer was demonstrated by the detection of other known crosslinked peptides and by the virtual absence of label in alpha 1CB7, CB8, and CB3. We feel that this simple methodological approach developed in these experiments will prove to be very useful in the analysis of collagen crosslinks present in insoluble collagens derived from normal tissues of various ages as well as from pathological states.

Animals↗