Tags for labeling protein N-termini with subtiligase for proteomics

The peptide ligase subtiligase, derived from subtilisin, has been employed in the identification of protein N-termini in complex mixtures. Here, the peptide ester substrates for the ligation reaction were optimized with respect to solubility, resulting in greater incorporation of the N-terminal tags. Additionally, the quantitation of the incorporated tags was explored, and a 'click' chemistry-based derivatization provided the ability to quantitate the tag to low nanomolar concentrations by sandwich ELISA. These new tags should expand the utility of subtiligase for the proteomic study of N-termini.     

Reductive methylation as a tool for the identification of the amino groups in alpha-bungarotoxin interacting with nicotinic acetylcholine receptor

alpha-Bungarotoxin (alpha Bgt) is a postsynaptic neurotoxin which blocks cholinergic transmission at the neuromuscular junction by binding tightly to the acetylcholine receptor (AcChR). The number of methylation sites in alpha Bgt has been shown to decrease significantly upon binding of the toxin to the AcChR [Soler, G., Farach, M. C., Farach, H. A., Mattingly, J. R., & Martinez-Carrion, M. (1983) Arch. Biochem. Biophys. 225, 872-878]. We have compared the chemical reactivities of amino groups in free and AcChR-bound alpha Bgt in an attempt to identify the regions in the alpha Bgt molecule that become masked upon binding to the AcChR. Free alpha Bgt and AcChR-bound alpha Bgt were reductively methylated with formaldehyde and sodium cyanoborohydride, and the rate of modification of each one of the available amino groups was followed by cleaving the methylated toxin with V8 protease and resolving the resulting peptides by reversed-phase, high-performance liquid chromatography. Under conditions of limited reagent availability, five of seven amino groups in free alpha Bgt reacted readily, whereas two other amino groups, probably those corresponding to Lys-51 and Lys-70, displayed lower reactivity. Upon binding to the AcChR, the rates of reductive methylation of residues Ile-1, Lys-26, and Lys-38 were considerably reduced (although to differing extents). The degree of protection was most pronounced for Lys-26. The rates of methylation of the amino groups in all other positions remained unchanged. These results allow further definition of the minimal binding surface of a representative neurotoxin.     

Modification of eremomycin by amine groups

Possible modification of eremomycin, a novel glycopeptide antibiotic by the amine groups with acylating agents such as Ac2O/MeOH and CH3(CH2)7COCl/Et3N and alkylating agents such as CH3CHO, NaBH and NaBH3CN was studied. N-Acetyl, N,N'-diacetyl. N-pelargoil, N-ethyl, N,N'-diethyl and N,N',N"-triethyl derivatives of eremomycin were prepared. Their structure was asserted and the order of the substitute introduction was determined. The antimicrobial activity against Bacillus subtilis and Staphylococcus aureus was assayed. It was found that with introduction of the ethyl substitutes to the eremomycin molecule the antibiotic activity lowered insignificantly whereas the acylation resulted in its decreasing by 1-2 orders.     

Quantification of primary amine groups available for subsequent biofunctionalization of polymer surfaces

Biocompatible polymers are commonly functionalized with specific moieties such as amino groups to modify their surface properties and/or to attach bioactive compounds. A reliable method is usually required to characterize amino group surface densities. In this study, aminated polyethylene terephthalate (PET) films were generated via an aminolysis reaction involving either ethylenediamine molecules (EtDA), in order to vary easily the amino group density on PET surfaces, or 25 kDa polyvinylamine (PVAm) as an alternative reagent preventing bulk damages resulting from the aminolysis reaction. Among commonly used dyes for amino group quantification, Orange II and Coomassie Brillant Blue (CBB) were selected to quantify the extent of amine grafting resulting from these derivatization procedures. Rapid and convenient colorimetric assays were compared to surface atomic compositions obtained from X-ray photoelectron spectroscopy (XPS) measurements. Orange II was found to be the most appropriate dye for quantifying primary amine groups in a reliable and specific way. Due to its unique negative charge and low steric hindrance compared to CBB, the Orange II dye was very sensitive and provided reliable quantification over a wide range of amino group surface densities (ca. 5 to at least 200 pmol/mm(2)). In order to further validate the use of the Orange II dye for amino group quantification, a heterobifunctional linker reacting with amino groups was then grafted on modified PET surfaces. Interestingly, the good correlation between the densities of adsorbed Orange II and covalently grafted linkers suggests that the Orange II method is a relevant, reliable, easy, and inexpensive method to predict the amount of amino groups available for subsequent functionalization of polymer surfaces.     

Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation

The colored complex formed between Cu+ and bicinchoninic acid is the basis of the bicinchoninic acid protein assay (P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B.J. Olson, and D.C. Klenk (1985) Anal. Biochem. 150, 76-85). Studies show that cysteine, tryptophan, tyrosine, and the peptide bond are capable of reducing Cu2+ to Cu+. Electrochemical studies and the magnitude of the color changes observed when the reaction is carried out at 37 degrees C indicate that tryptophan, tyrosine, and the peptide bond are not completely oxidized at this temperature. When the reaction temperature is increased to 60 degrees C, significantly more color formation is observed for these three groups. Studies with di-, tri-, and tetrapeptides and with proteins indicate that the extent of color formation is not the sum of the contributions of the individual color producing functional groups. Compounds with functional groups similar to those of cysteine, cystine, tyrosine, or tryptophan are shown to react with the bicinchoninic acid reagent. The color formed by these compounds in the presence of bovine serum albumin cannot be compensated for by using a reagent blank containing an identical concentration of the interfering compound.     

Parallel cascade identification as a means for automatically classifying protein sequences into structure/function groups

Current methods for automatically classifying protein sequences into structure/function groups, based on their hydrophobicity profiles, have typically required large training sets. The most successful of these methods are based on hidden Markov models, but may require hundreds of exemplars for training in order to obtain consistent results. In this paper, we describe a new approach, based on nonlinear system identification, which appears to require little training data to achieve highly promising results. Received: 16 March 1998 / Accepted in revised form: 2 June 1999     

Proteomics strategies for protein identification

The information from genome sequencing provides new approaches for systems-wide understanding of protein networks and cellular function. DNA microarray technologies have advanced to the point where nearly complete monitoring of gene expression is feasible in several organisms. An equally important goal is to comprehensive survey cellular proteomes and profile protein changes under different cellular states. This presents a complex analytical problem, due to the chemical variability between proteins and peptides. Here, we discuss strategies to improve accuracy and sensitivity of peptide identification, distinguish represented protein isoforms, and quantify relative changes in protein abundance.     

Chloroplast thylakoid membrane proteins having buried amine buffering groups

Some chloroplast thylakoid membrane proteins have anomalously low pK_a (near 7.8) amine groups, indicating that the buffering groups may be buried in hydrophobic regions and/or close to other positive charges. Other work has shown that the low pK_a amine group array is not in ready equilibrium with either the inner or outer bulk aqueous phases (Laszlo, J.A., Baker, G.M. and Dilley, R.A. (1984) J. Bioenerg. Biomembranes, 16, 37–51). Acetic anhydride reacts with the neutral amine and has been used as a probe for labeling the low pK_a amines. The buried array of buffering groups can be labeled with [³H]acetic anhydride in the dark only after the membranes were made leaky to protons with uncoupler addition. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis was used to separate the polypeptides and identify those that show the uncoupler-dependent labeling increase. Included in that group are polypeptides known to be associated with Photosystem II having M_r 17000, 22000 and 31000, some of the light-harvesting pigment proteins with M_r 24000–28000, the CF₀ component with M_r 8000 and some polypeptides associated with Photosystem I. A protein with M_r 15000 showed very large changes in labeling, but the identity of this polypeptide is unknown. The arrays of buried amine buffering groups are diversely distributed among membrane proteins and it is not clear what role, if any, they play in membrane function.     

Studies on sulfhydryl groups of Aspergillus niger amine oxidase.

Amino acid analysis of the amine oxidase of Aspergillus niger (monoamine:O2 oxidoreductase (deaminating), EC 1.4.3.4) showed a composition similar to that of bovine plasma enzyme. One molecule of enzyme contained 25 Cys residues. It was shown that 9 to 11 residues of Cys were titrated to be SH groups. The amine oxidase reaction was markedly inhibited by metal ions (Cu2+, Hg2+, Ag+). The enzyme was inactivated with SH reagents (phenyl mercuric acetate, Cl-HgBzO-) and the extent of this inactivation was dependent on the time of incubation with SH reagents. Also, the Cl-HgBzO- -inactivated enzyme was reactivated with cysteine and this reactivation was biphasic with the time of incubation. The Cl-HgBzO--inactivated amine oxidase was compared with the native enzyme in their reactivity with phenylhydrazine and their spectral properties. The results showed that the Cl-HgBzO--inactivated enzyme had lower reactivity with phenylhydrazine than the native enzyme and had higher absorbance values than the native enzyme around 400 nm wavelengths.     

Comparing hydrazine-derived reactive groups as inhibitors of quinone-dependent amine oxidases

Lysyl oxidase has emerged as an important enzyme in cancer metastasis. Its activity has been reported to become upregulated in several types of cancer, and blocking its activity has been shown to limit the metastatic potential of various cancers. The small-molecules phenylhydrazine and β-aminopropionitrile are known to inhibit lysyl oxidase; however, issues of stability, toxicity, and poorly defined mechanisms limit their potential use in medical applications. The experiments presented herein evaluate three other families of hydrazine-derived compounds – hydrazides, alkyl hydrazines, and semicarbazides – as irreversible inhibitors of lysyl oxidase including determining the kinetic parameters and comparing the inhibition selectivities for lysyl oxidase against the topaquinone-containing diamine oxidase from lentil seedlings. The results suggest that the hydrazide group may be a useful core functionality that can be developed into potent and selective inhibitors of lysyl oxidase and eventually find application in cancer metastasis research.     

Pyroglutamyl N-termini of thermal polyamino acids

It has been established indirectly that the N-termini of the thermal polyamino acids are pyroglutamic acid. This was determined by trifluoroacetic acid hydrolysis of the lactam ring followed by Dansyl labelling. The polyamino acids contained Ala, Gly, Glu, Leu, Phe, and Pro. In the experiments described here, the presence of pyroglutamic acid at the N-terminus of a polyamino acid was determined directly by the use of pyrrolidone carboxylyl peptidase. The enzyme catalyzes the removal of pyroglutamyl residues at the N-terminus of polypeptide chains. The polyamino acids used in these studies contained glutamic acid, aspartic acid, alanine, glycine, isoleucine, proline and valine. Alkaline hydrolysis was also used to determine indirectly that the N-termini of these polyamino acids are pyroglutamic acid. Another interesting finding was that many of the amino acids in the polymerization mixture were found to occur penultimate to the N-terminal amino acid. This is interpreted to mean that the diffusible fraction contains many polyamino acids.     

Chemical modification of amine groups on PS II protein(s) retards photoassembly of the photosynthetic water-oxidizing complex

Four Mn atoms function as catalysts in the water-oxidizing complex located on the oxidizing side of PS II. We have studied the involvement of amine groups of the PS II proteins in photoligation of Mn2+ to the apo water-oxidizing complex, using the combined techniques of photoactivation and chemical modification with the modifiers methyl acetimidate (MAI), acetic acid N-hydroxysuccinimide ester (NHS), and 2,4,6-trinitrobenzenesulfonic acid (TNBS). Chemical modification of hydroxylamine-treated PS II core complexes decreased their capacity for restoration of oxygen evolution and photoligation of Mn2+ to the apo water-oxidizing complex (WOC), but did not affect their electron transfer activity in the vicinity of PS II. The number of functional high-affinity Mn-binding sites, but not of low-affinity sites, was significantly modulated by chemical modification. Kinetic analysis of photoactivation with the repetitive flashes revealed that the intermediate generated during a photoactivation process was destabilized by the chemical modification. To identify which proteins possess the amine groups involved in ligation of functional Mn, we examined the difference in NHS biotinylation between PS II core complexes with and without the Mn cluster. NHS biotinylation resulting in altered ligation of functional Mn apparently occurred on three proteins: an antenna chlorophyll binding protein (CP47), a light-harvesting chlorophyll protein (CP29), and another chlorophyll binding protein (PS II-S). Of these proteins, only the Mn-dependent biotinylation of CP47 was found to occur independently of the application of an NHS-masking concentration before removal of the functional Mn. These results suggest that lysyl residues of CP47, and perhaps also CP29 and PS II-S, function in direct photoligation of Mn2+ to the apo WOC.     

Using DCMU-fluorescence method for the identification of dominant phytoplankton groups

For the identification of ecologically significant dominant groups of phytoplanktonic algae a polychromatic DCMU-induced fluorescence method is recommended. A special fluorometer equipped with a system of replaceable filters is used to differentiate three regions of the spectrum (410 ± 20, 510 ± 20 and 540 ± 10 nm) that can excite the basic light-harvesting pigments. Total and differential (for every algal taxon studied) chlorophyll a calculated from the fluorescence signals is in good agreement with biomass estimates from direct cell counts for several different trophic types of aquatic systems. This is made possible by the vizualization of the ratios of fluorescence signal values in their own coordinates: first, to decide whether it is necessary to correct linear equations in order to eliminate negative solutions; second, to determine the possibility of nulling the negative solution if a point is situated close to a side of the triangle; and third, to reduce the number of linear algebraic equations to two if the points are situated along one of the triangle sides or to one if the points are gathered at the apex. The polychromatic DCMU-induced fluorescence method can be used for monitoring natural phytoplankton populations to detect changes in their taxonomic structure.     

Cleavable ester-linked magnetic nanoparticles for labeling of solvent-exposed primary amine groups of peptides/proteins

To study the solvent-exposed lysine residues of peptides/proteins, we previously reported disulfide-linked N-hydroxysuccinimide ester-modified silica-coated iron oxide magnetic nanoparticles (NHS–SS–SiO₂@Fe₃O₄ MNPs). The presence of a disulfide bond in the linker limits the use of disulfide reducing agent during protein digestion and allows unwanted disulfide formation between the MNPs and protein. In the current work, the disulfide bond was replaced with a cleavable ester group to synthesize NHS ester-modified SiO₂@Fe₃O₄ MNPs. Use of the cleavable ester group provides an improved method for protein labeling and allows the use of disulfide reducing agents during protein digestion.     

UGUS, a reporter for use with destabilizing N-termini.

Due to constraints in vector construction, reporter polypeptides often carry N-terminal sequences of extraneous origin. Since protein half-life can be influenced by small determinants in the N-terminus, such foreign sequences can destabilize proteins and compromise results of reporter-based studies. We provide a real-life example of this problem (destabilizing sequences derived from a ribosomal protein) and show that it can be solved with the ubiquitin fusion technique, in which ubiquitin sequences are placed upstream of the reporter, in our case beta-glucuronidase. Post-translational processing by characterized pathways removes the ubiquitin together with destabilizing sequences, generating a stable reporter whose N-terminus is constant.     

Colorimetric artificial tongue for protein identification

Artificial tongue systems are multisensory devices which are highly desirable for the analysis and recognition of complicated composition samples. Herein, a low-cost and simple colorimetric sensor array for identification and quantification of proteins were reported. Using prophyrin, porphyrin derivatives (mainly metalloporphyrins) and chemically responsive dyes as the sensing elements, the developed sensor array of artificial tongue showed a unique pattern of colorific change upon its exposure to proteins. The composite pattern for each sample was subjected to principal component analysis (PCA), thus providing a clustering map for more practical visualization. All the pure and mixed proteins, as well as denatured proteins, gave distinct patterns, thus resulting in their unambiguous identification. The PCA analysis also suggested that the unique pattern of colorific change may be due to the change of protein conformation and local environmental pH. These results demonstrate that the developed colorimetric artificial tongue system is an excellent sensing platform for identification and quantitative analysis of protein samples.