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Peptide maps were prepared by high-voltage electrophoresis and chromatography for t 30 degrees C mutant and the original strain A Port Chalmers 1/73 H3N2 of influenza virus. Clear differences in the distribution and number of spots between the mutant and the original strain were found. The differences are due presumably to changes in the amino-acid sequences.
Tryptic peptide map analyses of five K- and three D-gene products of various H-2 haplotypes are presented. These data support earlier sequence studies and demonstrate that the variations in allelic gene products of the K or D loci are scattered throughout much of the polypeptide chains. Furthermore, the K allelic gene products are no more closely related to one another than they are to the D allelic gene products. This apparent lack of K-ness and D-ness places interesting constraints on the genetic organization and evolutionary history of the genes encoding the transplantation antigens.
We have examined and optimized several parameters to generate efficient, high-resolution, high-information tryptic peptide maps of monoclonal antibodies and their Fab fragments, without separating the H and L chains, using reversed-phase high-pressure liquid chromatography. Use of a high protease-to-substrate ratio with optimized digestion time and HPLC gradient conditions led to a reproducible mapping of the reduced, carboxymethylated Fab fragments of two antibodies. The technique was then used to screen Fab lots for batch-to-batch consistency, and for examining the effect of 10 mM cysteine on papain cleavage of whole antibody. The technique was modified by labeling cysteine with chromophoric analogues of iodoacetamide instead of radiolabeled iodoacetamide, resulting in a three-dimensional peptide map. With multiwavelength detection, this consisted of simultaneous observation of all chromophores at 214 nm, those with aromatic residues at 280 nm, and those with cysteine at 422 nm, without collecting and counting each peak to identify cysteine-containing peptides.
The small GTP-binding protein (G protein) Rac1 is an obligatory participant in the assembly of the superoxide (O2-.)-generating NADPH oxidase complex of macrophages. We investigated the effect of synthetic peptides, mapping within the near carboxyl-terminal domains of Rac1 and of related G proteins, on the activity of NADPH oxidase in a cell-free system consisting of solubilized guinea pig macrophage membrane, a cytosolic fraction enriched in p47phox and p67phox (or total cytosol), highly purified Rac1-GDP dissociation inhibitor for Rho (Rho GDI) complex, and the activating amphiphile, lithium dodecyl sulfate. Peptides Rac1-(178-188) and Rac1-(178-191), but not Rac2-(178-188), inhibited NADPH oxidase activity in a Rac1-dependent system when added prior to or simultaneously with the initiation of activation. However, undecapeptides corresponding to the near carboxyl-terminal domains of RhoA and RhoC and, most notably, a peptide containing the same amino acids as Rac1-(178-188), but in reversed orientation, were also inhibitory. Surprisingly, O2-. production in a Rac2-dependent cell-free system was inhibited by Rac1-(178-188) but not by Rac2-(178-188). Finally, basic polyamino acids containing lysine, histidine, or arginine, also inhibited NADPH oxidase activation. We conclude that inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of certain small G proteins is not amino acid sequence-specific but related to the presence of a polybasic motif. It has been proposed that such a motif serves as a plasma membrane targeting signal for a number of small G proteins (Hancock, J.F., Paterson, H., and Marshall, C.J. (1990) Cell 63, 133-139).
A peptide mapping technique is described which uses a two dimensional format to display CNBr peptides of collagen chains. Biosynthetically-labeled products from 250,000 cells are analyzed in each map by a rapid procedure which does not require preliminary purification steps. Proteins trapped within polyacrylamide gels are digested with CNBr under conditions where diffusion of radiolabeled peptides from gels is negligible, and the reaction products are recovered quantitatively by electroelution. Peptide maps of pro alpha chains, alpha chains, and TCA chains cleaved with mammalian collagenase are presented with the identification of specific fragments. This method is useful for the analysis of structural abnormalities in collagen proteins from patients with certain genetic disorders, examination of collagenous proteins produced by primary cultures which exhibit phenotypic switching, and identification of new collagen types.
We present an approach that allows matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) peptide mapping of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (NC). After blocking the nitrocellulose membrane with polyvinylpyrrolidone-40 the immobilized proteins are visualized using India Ink staining which allows the detection of low nanogram amounts of protein. The utilization of a low concentration of Tween 20 (0.05%) in the India Ink staining solution does not negatively impair the quality of the mass spectra. Due to the virtual nondestructive nature of the stain proteolytic peptides could be recovered from the NC membrane. Taking into account minor precautions during the sample manipulation and concentration and by loading the sample onto a pre-crystallized matrix layer, high quality mass spectral data were obtained on <100 femtomoles of protein loaded onto the gel. Finally, the use of India Ink in conjunction with Western blot analysis is also demonstrated. A rat plasma protein, characterized by Western blot as a covalently modified protein-drug compound, was subjected to peptide mapping and post source decay (PSD) sequencing of peptides. The zomepirac-modified protein was identified as the alpha-subunit of fibrinogen.
Four isoperoxidases of turnip root and isoperoxidase C of horseradish root were digested with trypsin, and their peptide maps, prepared by high-voltage paper electrophoresis, were compared. All five tryptic digests were completely soluble at pH 8. The maps were developed with a variety of general and specific reagents: ninhydrin, histidine, tyrosine, tryptophan and arginine reagents. Cystine peptides and cysteic acid derivatives have also been characterized. All detected half-cystine residues seemed engaged in disulfide bridges. For each individual peroxidase the number of specifically staining peptides agreed very well with the amino acid composition. The two most acidic peroxidases of turnip, P1 and P2, only differ significantly in one peptide. The P2 gene is tentatively proposed to have developed from the P1 gene by a single base mutation, changing an asparagine residue to alysine residue. A less acidic turnip peroxidase, P3, is distinct, although related to peroxidases P1 and P2. Horseradish isoperoxidase C also belongs to this group which appears to be closely related in the amino acid sequences around four disulfide bridges. Peroxidase P7 differs from this group, at least around two of its disulfide bridges, and therefore, may differ from the other four in parts of its three dimensional structure. Sequences of particular importance to peroxidase function must be present in all peroxidases. From the peptide mapping studies we only find two highly homologous sequences present in all five examined peroxidases. Both contain histidine. This finding corroborates previous suggestions of two histidine sequences near the peroxidase heme prosthetic group. The rules applied in relating peptides of different proteins are outlined, and the sources of errors in mapping of glycoproteins of high carbohydrate content (about 20%) are discussed in detail.
A nanoliter enzyme microreactor was developed for on-line capillary electrophoresis (CE) peptide mapping of proteins, allowing picomole quantities of proteins to be digested. The enzyme microreactor was formed by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary. Highly efficient digestion of three protein standards was demonstrated. The detection of peptide fragments in CE was enhanced by post-column derivatization and laser-induced fluorescence detection. The microreactor has a volume of about 30 nL and is coupled with a separation capillary via a fluid joint for on-line digestion. The overall analysis, including digestion and separation, lasted only about 16 min. Column efficiencies > 300 000 plates/m were obtained for most peaks in the electropherogram of an on-line peptide mapping experiment of denatured alpha-lactalbumin under optimal conditions.
Short columns, packed with pellicular sorbents made of 2-micron fluid-impervious silica microspheres, were used at elevated column temperatures for rapid peptide mapping by high-performance liquid chromatography (HPLC). Enzymic digests of various proteins were chromatographed by gradient elution. In many cases the time of analysis was 10 min or less. In order to increase the retention particularly, that of short, polar peptides under such conditions, 1 mM octyl sodium sulfate or 5 mM hexyl sodium sulfate were added to the starting eluent. The length of the 4.6 mm I.D. columns was 30 or 75 mm, the sample load was in the range of 10-1000 pmoles. Highest analytical sensitivity was obtained at a flow-rate of 0.5 ml/min and room temperature, whereas for rapid analysis flow-rates of up to 2 ml/min were used at 80 degrees C. This temperature allowed the use of relatively high flow velocities of the mobile phase without significant loss in efficiency. The method was highly reproducible, as shown by the results obtained by automated analysis of cyanogen bromide fragments of lysozyme at high speed. The quality of the rapid peptide maps compares favorably with that of maps obtained by standard reversed-phase HPLC methods, which require much longer analysis times.
Proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained in situ with either 5-(dimethylamino)-1-naphthalene sulfonyl chloride (dansyl chloride) or fluorescein isothiocyanate. This staining procedure can be carried out in less than 30 min without previous fixation of the proteins. It is not dependent on such factors as charge or molecular weight of the proteins and can detect 50 ng of protein in a 10-mm-wide gel slot. Fluorescent staining with dansyl chloride was used to localize proteins after electrophoresis for subsequent electroelution, amino terminal analysis, and peptide mapping. The electroelution can be carried out in less than 3 h with yields approaching 100%. The staining of only one strip of a preparative gel allowed the electroelution of proteins without covalent modification. For amino terminal analysis, identical results were obtained when the hydrolysis step was carried out after electroelution or directly in the gel pieces. The peptide mapping can be carried out with the proteins in solution (after electroelution) or directly in the gel pieces. The amino terminal and peptide mapping analysis of each protein in a mixture can be completed within 30 h from the beginning of the electrophoretic fractionation. The method appears to be applicable to a wide range of proteins showing very different biochemical properties.
Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively.
A method for simultaneous peptide mapping of polypeptides contained in a mixture is presented. The polypeptides were first separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The strip of gel containing these unstained polypeptide bands was subsequently embedded perpendicular to the direction of electrophoresis in the stacking gel of a second gel. The proteolytic enzymes, loaded on top of the second gel, were brought in contact with the substrates through moving boundary electrophoresis. The peptides thus generated were then resolved by electrophoresis in a gradient gel. A polychromatic silver staining method added an extra dimension to the identification and characterization of the peptides in the maps obtained in that specific peptides got specific colors. Moreover, the sensitivity of this method was illustrated by the demonstration that original quantities in the submicrogram range of nonradioactive proteins (exemplified here by the structural proteins of densonucleosis virus) largely sufficed for satisfactory maps. Other advantages of this procedure over current methods included (i) the elimination of the purification step (and consequently virtually no loss or contamination), (ii) that only the strict minimum of material (necessary for the ultimate visualization of the maps) had to be used, (iii) that no special two-dimensional electrophoresis equipment was needed, and (iv) the consistency, speed, and simplicity of the method.
An automatic computer technique was used to compare the retention times and ultraviolet spectra of sixty-two peaks in peptide maps of three monoclonal antibodies against human tumor necrosis factor (TNF) and one monoclonal antibody against recombinant factor VIII. The anti-TNF monoclonal, B6, which has an overlapping epitope with the anti-TNF monoclonal, A10G10, had a 90% peak match with A10G10. The anti-TNF monoclonal, A6, with a different epitope to TNF than A10G10, had only a 60% peak match. The A6 match to A10G10 was similar to the 50% peak match of the anti-factor VIII monoclonal with A10G10. The results of this study suggest that peptide mapping can be used as a quantitative characterization technique for comparing monoclonal antibodies.
Two protein kinases of Mr 43,000 and 23,000 from yeast, belonging to type-1 casein kinases, were purified to apparent homogeneity and used for investigation of their immunological affinity and for comparison of their peptide map patterns. The results obtained showed that antibodies against the 43 kDa kinase did not react with the 23 kDa enzyme. Moreover, the peptide maps of the radioiodinated kinases obtained either by chemical cleavage of peptide bonds in the presence of CNBr or by a limited digestion with V8 protease were completely different. All these observations point to the lack of relatedness between the two investigated enzymes.
The basidiomycete Trametes versicolor, a white-rot fungus and potent degrader of lignin, produces multiple forms of extracellular peroxidases. Nine of these forms, six lignin peroxidases and three manganese(II) peroxidases, purified as described in the preceding paper, were characterized by amino-terminal sequencing, amino acid analyses, carbohydrate analyses, or peptide mapping. For two of the lignin peroxidase forms, tryptic peptides were isolated and sequenced to an extent corresponding to about 40 and 30%, respectively, of the primary structure. Eight of the nine peroxidases investigated were found to possess unique amino-terminal regions. A comparison of the sequences shows 57% of the residues to be identical, indicating a common ancestry for the lignin peroxidase and the manganese(II) peroxidase. The degree of identity among the five lignin peroxidases is about 80% and among the three manganese(II) peroxidases about 70%. Pairwise comparisons of the sequences disclosed that some of the lignin peroxidases are very closely related, either identical or differing only in a single amino acid residue of the thirty-five investigated. These close relationships are also supported by peptide mapping and by similarities in amino acid compositions. Tyr is absent in all isozymes. Lignin and manganese(II) peroxidases showed the presence of glucosamine and mannose in an amount corresponding to 3 to 6% of the molecular mass of the proteins. The carbohydrate compositions are compatible with the presence of 1, 2, and 3 sites of N-glycosylation. The results obtained strongly suggest that the complexity in the peroxidase pattern displayed by the fungus (T. Johansson and P.O. Nyman, Arch. Biochem. Biophys. 300, 49-56, 1993) can largely be accounted for by a heterogeneity at the gene level, probably in the form of multiple structural genes. Two recently published genes from genomic clones of T. versicolor are identical in sequence to two of the lignin peroxidases characterized here.
A structural homology is established between three DNA-binding phosphoproteins located in the 42 to 44 kDa range, referred to as pp42, pp43 and pp44, from Chironomus tentans salivary gland cells by in situ peptide mapping. The staining patterns of pp42, pp43 and pp44 which resulted from digestion with Staphylococcus aureus V8, trypsin or papain proteases show the presence of 8 to 15 spots majority of which have identical mobility. In the patterns of the digests generated by treatments with trypsin about 10 spots appear in common between any pair of the protein substrates. In addition, each pattern includes two to three peptides of mobility not present in the other. Thus the peptide mapping of pp42, pp43 and pp44 based on the staining patterns of proteolytic digests suggest the existence of structural homology between the three unlabelled substrates. The proteolytic peptides carrying the rapidly turning over phosphate groups form markedly different electrophoretic patterns than the unlabelled peptides visualized by staining. Treatment of 32P-labelled pp42, pp43 and pp44 with V8 generates only one labelled fragment in the 30 kD range. The cleavage patterns of pp44 produced by chymotrypsin or papain contain seven to ten labelled fragments while those of pp42 and pp43 contain only two. The 32P-labelled tryptic peptides of pp42, pp43 and pp44 exhibit a ladder pattern for each substrate which probably arise by a consecutive removal of 25 to 35 amino acid residues from the primary digestion products pp29, pp29.5 and pp30 by cleavage of four to five putative interdomain regions. The possibility that these three structurally related phosphoproteins belong to the category of transcription factors is discussed.
We describe an approach that utilizes nonlinear peptides to direct the assembly of previously reported Self-Assembling Fibers (SAFs). The SAF system comprises two complementary linear peptides, SAF-p1 and SAF-p2a, which combine to form exclusively linear, nonbranched fibers. The Matrix-Programming (MaP) peptides described herein are based on these peptides: they comprise two or three half-peptide blocks derived from the SAF peptides, which are conjugated via dendritic hubs. Different MaP peptides coassembled with the standard SAF peptides to form specific structures, such as hyperbranched networks, polygonal matrices, and regularly segmented and terminated fibers. The role of each half-peptide block in dictating the different features has been elucidated. This provides a strong basis for designing new peptide-based nanostructured materials from the bottom up.
The conditions for tryptic digestion and subsequent peptide mapping of the ATP-dependent proteolysis cofactor ubiquitin and its derivatives are described. In aqueous solution, the native ubiquitin which is composed of 76 amino acids undergoes only a single cleavage at arginine-74. Full digestion of ubiquitin was obtained in 6.5 M urea, although cleavages at lysine-33 and arginine-74 were slow. Peptide mapping was achieved by reverse-phase high-performance liquid chromatography with a C18 column using a trifluoroacetic acid/triethylamine buffer system and acetonitrile as eluants. The peptides, separated using a linear gradient, were identified by amino acid analysis. Derivatives analyzed by this method include oxidized, monoiodotyrosyl, and diiodotyrosyl ubiquitin. This technique will be useful in examining peptides of chemically modified ubiquitin with respect to extent and specificity of modification. In addition, this technique will be useful in comparing ubiquitin peptides of different organisms.