An improved procedure for high-sensitivity microsequencing: Use of aminoethyl aminopropyl glass beads in the beckman sequencer and the Ultrasphere ODS column for PTH amino acid identification

An improved procedure for high-sensitivity microsequencing is described. The method employs (i) aminoethyl aminopropyl glass beads for in situ purification of quadrol by absorbing α-amino group reactive impurities commonly found even in highly purified quadrol and (ii) an Altex 5-μm Ultrasphere ODS column and highly purified methanol which is available commercially to identify phenylthiohydantoin derivatives of amino acids by high-pressure liquid chromatography. The efficiency of the method has been demonstrated by the results of sequence analyses of peptides obtained by acid and cyanogen bromide digestions of viral proteins. A significant increase in the initial and repetitive yields has been consistently observed by this procedure.     

Solid-phase sequencing on the gas-phase sequencer

Automated Edman degradation has been successfully used for determining the primary structure of numerous peptides and proteins. Quantitative solid-phase Edman degradation has great potential use for amino acid sequence analysis of synthetic peptides assembled on resin support by the Merrifield procedure. We report here the combined use of a modified gas-phase sequencer program and our improved reversed-phase HPLC analysis for PTH-amino acids to carry out the sequence analysis on synthesized peptide resins. This approach is far more sensitive than using glass beads on the conventional solid-phase sequencer. The peptide was assembled on copoly (styrene-1% divinylbenzene) resin beads at an initial substitution of 0.54 mmol/g. On a routine basis, 10-15 resin beads are used, and a repetitive yield of 94% is obtained: as few as 4 beads can be successfully sequenced. The HPLC PTH-amino acid analysis is sensitive down to subpicomole quantities. This procedure offers a sensitive and rapid analytical tool for checking the purity of peptides as they are being assembled on solid support.     

Microanalysis of the amino-acid sequence of monomeric beta-lactoglobulin I from donkey (Equus asinus) milk. The primary structure and its homology with a superfamily of hydrophobic molecule transporters

The complete primary structure of donkey beta-lactoglobulin I was determined by pulsed-liquid phase microsequencing of tryptic peptides. The protein has been isolated in monomeric form and it corresponds to monomeric beta-lactoglobulin of type I. With the inclusion of donkey beta-lactoglobulin I there are 13% common residues amongst the members of the beta-lactoglobulin family. Donkey beta-lactoglobulin I is homologous to the retinol-binding protein, bilin-binding protein and five other proteins belonging to the new superfamily of hydrophobic molecule transporters. A rapid method for peptide isolation and the strategy for microsequencing of this protein have been described.     

Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord

A new peptide, designated "neuromedin K" has been discovered and isolated from porcine spinal cord by using bioassays for a tachykinin-like effect on the contractility of smooth muscle preparation from guinea-pig ileum. Porcine neuromedin K has been identified by microsequencing as: Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. The sequence thus determined has been confirmed by synthesis. Neuromedin K has been found to have not only a remarkable sequence homology to kassinin and substance P, but also a prompt stimulant activity on guinea-pig ileum in a manner similar to that of substance P, suggesting that neuromedin K may be involved in neural transmission.     

Identification and immunocytochemical localization of proctolin in pericardial organs of the shore crab, Carcinus maenas

The occurrence of proctolin (Arg-Tyr-Leu-Pro-Thr) in crab neurohemal pericardial organs (POs) has been demonstrated by isolation of the pentapeptide by HPLC and manual microsequencing according to the DABITC-PITC double coupling technique. From one pair of POs approximately 5.4 pmol were obtained (= 45 pmol/mg protein). Immunocytochemically, an extensive system of positive structures was found in both whole mount preparations and semithin sections, consisting of numerous varicose fibres of varying diameter and many knoblike neurosecretory terminals abutting upon the epineurium of the PO trunks. The relatively high concentration in the POs as well as the pattern of proctolin-positive fibres and terminals clearly suggest a neurohormonal role of the pentapeptide in decapod crustaceans.     

Characterization of a Borna disease virus glycoprotein, gp18.

Borna disease virus is a nonsegmented negative-strand RNA virus that causes neurologic disease in a wide variety of animal hosts. Here we describe identification and characterization of the first glycoprotein in this viral system. The 18-kDa glycoprotein, gp18, has been purified from infected rat brain. Isolation and microsequencing of this protein allowed identification of a 16.2-kDa open reading frame in the viral antigenome. Lectin binding and endoglycosidase sensitivity assays indicate that gp18 is an unusual N-linked glycoprotein.     

Isolation and complete primary sequence of a new ovine wild-type beta-lactoglobulin C

A new wild type of beta-lactoglobulin has been identified in the milk of sheep. It has been designated as ovine beta-lactoglobulin C. Its primary structure has been determined by direct protein microsequencing of intact protein and RP-HPLC-derived tryptic peptides. The new beta-lactoglobulin C is a subtype of ovine beta-lactoglobulin A with a single exchange Arg-Gln at position 148. This exchange may influence polymerisation of beta-lactoglobulin since in the crystal structure of orthorhombic bovine beta-lactoglobulin, residues 145-150 constitute a short beta-sheet region involved in dimer formation by pairing of dyad-related strands.     

A home-made solid-phase sequencer operating at the nanomole level

The bulding and functioning of a fully automated solid-phase sequencer is described. The peptide is coupled via its -carbonyl end to activated glass beads and successively reacted with Chang's and Edman's reagents. All operations are electronically controlled by the automated programmer. All components necessary to build the machine are commercially available. This sequencer has been used at a nanomole level in the final phase of a protein sequence determination. The overall cost as well as the sensitivity and efficiency of the final product compare favourably to those of commercial machines.     

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated M _r of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.     

Purification and characterization of natural Ara h 8, the Bet v 1 homologous allergen from peanut, provides a novel isoform

The peanut allergen Ara h 8 is an important allergen for birch pollen allergic patients because of the cross-reactivity to the homologous Bet v 1. As the existence of Ara h 8 has been shown at the cDNA level so far (AY328088) and the allergen has indirectly been detected as natural protein, it was the aim of our study to identify natural Ara h 8 in peanut extract and to develop a purification strategy. This was achieved using a unique combination of purification steps, including optimized extraction conditions, size exclusion and ion exchange chromatography and treatment of the interfering contaminants with iodoacetic acid. A characterization of the protein by microsequencing showed discrepancies to the deduced amino acid sequence of AY328088. For this reason, we cloned and expressed a new Ara h 8 isoform from cDNA (EU046325). This IgE-reactive protein corresponds to the results of microsequencing, ESI-FTICR-MS and trypsin fingerprinting analysis of the authentic and purified nAra h 8. Apart from the ultimate use of recombinant allergens for diagnostic procedures, there is also a scientific need for the natural counterpart, as it represents an excellent reference point by which to compare protein characteristics and to standardize diagnostic and therapeutic allergens.     

Sequence determination of eglin c using combined microtechniques of amino acid analysis,peptide isolation,and automatic Edman degradation

The complete amino acid sequence of a proteinase inhibitor, eglin c (M_r 8100), has been determined with less than 150 μg of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothyiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.     

Typing single-nucleotide polymorphisms using a gel-based sequencer

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot™ kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotype (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.     

Purification and complete amino acid sequence of alpha-human atrial natriuretic polypeptide (alpha-hANP)

The present survey for natriuretic factors in human atrial extract was performed by using in vitro assay for the relaxant effect on the contractility of chick rectum. Three distinct components (alpha, beta and gamma) of a potent relaxant activity were found in the chromatographic regions of the crude extract. As alpha-component of Mr 3,000 daltons, a 28-amino acid peptide has been isolated in a pure state and found to elicit potent diuretic and natriuretic activities as well as vasorelaxant activity, when injected into the assay rats. Accordingly, we proposed a name "alpha-human atrial natriuretic polypeptide (alpha-hANP)" for the peptide. The complete amino acid sequence of the peptide has been established by microsequencing as well as synthesis.     

Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.     

Metal Chelates as Reversible Stains for Detection of Electroblotted Proteins: Application to Protein Microsequencing and Immunoblotting

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ferrous complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.     

Chemical modification of peptides by hydrazine.

High pressure ('performance') liquid chromatography on reverse-phase supports has been used to characterize the products arising from the hydrazine treatment of peptides. In addition to converting arginine residues into ornithine, the reaction was found to cleave predominately Gly-Xaa, Xaa-Gly, Asn-Xaa and Xaa-Ser peptide bonds. Peptide-bond cleavage and deguanidation was studied as a function of time of exposure to hydrazine, hydrazine concentration and temperature. The convenience of this method of chromatography for the rapid low-cost separation and isolation of peptides, as well as their reaction products, is illustrated at the level of material required for solid-phase microsequencing.     

Expression and purification of functional human anthrax toxin receptor (ATR/TEM8) binding domain from Escherichia coli

Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. Anthrax receptors play a crucial role in the pathogenesis of the anthrax disease. Anthrax toxin receptor ATR/TEM8 VWA domain is responsible for the binding of protective antigen (PA) of B. anthracis, and thus an attractive target for structure-based drug therapies. However, the production of soluble and functional ATR/TEM8 VWA domain currently requires the use of mammalian expression systems. In this work, we expressed the ATR/TEM8 VWA domain as a fusion protein in Escherichia coli. Recombinant ATR/TEM8 VWA domain has been purified to homogeneity, and its identity has been verified by both N-terminal protein microsequencing and mass spectrometry. The purified ATR/TEM8 VWA domain exhibits very high affinity to PA based on BIAcore assay. Moreover, like the domain expressed in mammalian system, the bacterially expressed ATR/TEM8 VWA domain can block cytotoxicity induced by anthrax toxins, suggesting that the bacterially expressed ATR/TEM8 VWA domain is properly folded and fully functional.     

Isolation and identification of human brain natriuretic peptides in cardiac atrium

By using a radioimmunoassay (RIA) system newly established for human brain natriuretic peptide (BNP), a high concentration of immunoreactive (ir-) human BNP (hBNP) has been found in cardiac atrium (1). Two molecular forms of ir-hBNP of 4K and 13-15K were isolated from atrial extracts by using anti-hBNP IgG immunoaffinity chromatography and reverse phase high performance liquid chromatography (HPLC). By microsequencing, the peptides were determined to be a pro-hBNP (gamma-hBNP) and its C-terminal 32-amino acid peptide (hBNP-32). Based on these results, in cardiac atrium, hBNP is found to be processed in a pathway similar to that of porcine BNP (pBNP) but distinct from that of rat BNP, although low MW hBNP-32 is a major form in contrast to pBNP which exists as a high MW gamma-pBNP.     

Comparative gas phase and pulsed liquid phase sequencing on a modified Applied Biosystems 477A sequencer

A simple and inexpensive modification of the Applied Biosystems 477A sequencer, to run in the pulsed liquid-phase and in the gas-phase mode of the Edman chemistry, is described. This modification is especially useful for sequencing samples on polyvinylidene difluoride (PVDF) membranes. Additional carriers are required if a sample on a PVDF membrane is sequenced with the pulsed liquid-phase degradation program of the 477A. In the gas-phase mode no such carriers are needed. This eliminates time-consuming preconditioning sequencer cycles and reduces the sequencer background. In addition, initial coupling yields in the gas-phase mode exceeded those in the pulsed liquid-phase mode, whereas the average repetitive yields were similar. Samples spotted onto glass fiber filters pretreated with polybrene and samples spotted or electroblotted onto PVDF membranes were examined. A number of advantages of the gas-phase mode are presented.