Primary structure of Paim I, an alpha-amylase inhibitor from Streptomyces corchorushii, determined by the combination of Edman degradation and fast atom bombardment mass spectrometry

Paim I, a protein alpha-amylase inhibitor, inhibits animal alpha-amylases from pig, dog, cow, horse, etc. but has no activity against human salivary and pancreatic amylases. The primary structure of Paim I has been determined by Edman degradation and fast atom bombardment mass spectrometry (FABMS). This protein is a single-chain polypeptide of 73 amino acid residues with a calculated molecular weight from the sequence data of 7415.3 (monoisotopic molecular weight) and 7420.2 (average molecular weight). The sequencing strategy chosen for Paim I consists of four steps. First, the accurate molecular weights of the intact and tetra-S-carboxymethylated Paim I are determined by fast atom bombardment mass spectrometry. Second, the primary fragments generated by Staphylococcus aureus V8 protease are isolated by reversed-phase high-performance liquid chromatography. The molecular weights of these subpeptides are determined by FABMS. The peptides that must be sequenced are selected by the molecular weights of these subpeptides and the tetra-S-carboxymethylated Paim I. Third, these subpeptides and the whole protein are sequenced by automated Edman degradation. Finally, the primary structure of tetra-S-carboxymethylated Paim I is confirmed by the combination of tryptic, chymotryptic, and S. aureus V8 protease digestion and FABMS. The sequence of Paim I is compared with those of Haim II, Hoe-467A, Z-2685, and AI-3688 because they have different alpha-amylase inhibition spectra against mammalian alpha-amylases but belong to a family of related proteins.     

The primary structure of thioredoxin from Chromatium vinosum determined by high-performance tandem mass spectrometry

The primary structure of thioredoxin, a redox protein isolated from Chromatium vinosum, was determined by high-performance tandem mass spectrometry, which permitted sequencing of the 14 peptides (ranging in length from 2 to 18 amino acids) generated by digestion with trypsin and of several peptides produced by Staphylococcus aureus protease. The mass spectrometrically determined molecular weights of the peptides from the latter digest were used to properly align the tryptic peptides, which could also be accomplished on the basis of the considerable homology with Escherichia coli thioredoxin. Finally, the molecular weight of the Chromatium thioredoxin was determined by mass spectrometry and found to be 11,748.0, in good agreement with 11,750.2 calculated for the proposed sequence. Although it was difficult to establish by mass spectrometry, five leucines and three isoleucines could be identified, leaving only eight undifferentiated.     

Purification and primary structure of proteinous alpha-amylase inhibitor from Streptomyces chartreusis.

A new polypeptide inhibitor, AI-409, that inhibits human salivary alpha-amylase, was purified from a fermentation broth of Streptomyces chartreusis strain No. 409. This protein consists of a single-chain polypeptide of 78 amino acid residues, and includes two disulfide bridges. The primary structure of AI-409 and the locations of the disulfide bridges were identified by enzymatic digestion and the automatic Edman technique. Enzymatic digestion was done with trypsin, carboxypeptidase Y, and chymotrypsin. One of the disulfide bridges was between Cys(10) and Cys(26), and the other between Cys(44) and Cys(71).     

The primary structure of human EGF produced by genetic engineering, studied by high-performance tandem mass spectrometry

The primary structure of human epidermal growth factor (hEGF), which was produced by Escherichia coli using recombinant DNA technique, has been studied by tandem mass spectrometry. The molecular weight of hEGF (about 6200 amu) was determined by fast atom bombardment mass spectrometry. Then reduced and carboxymethylated hEGF was digested by chymotrypsin into seven peptides which could cover the whole sequence of hEGF. The amino acid sequences of five of these seven peptides could be confirmed by tandem mass spectrometry with or without isolation by high-performance liquid chromatography (HPLC). After isolation by HPLC, the other two peptides were digested with trypsin or thermolysin into small peptides, and sequenced by tandem mass spectrometry.     

Isolation and structure elucidation of an alpha-amylase inhibitor, AI-3688, from Streptomyces aureofaciens

A novel polypeptide inhibitor, AI-3688, which acts upon human pancreatic alpha-amylase, was isolated from fermentation broth of Streptomyces aureofaciens. The purified peptide contains no unusual amino acids. Its Mr is 3936. The primary structure of AI-3688 was elucidated by automatic Edman degradation of the native or modified inhibitor. Two intramolecular cysteines form a disulphide bridge, thus creating a ring structure consisting of 17 amino acids. Strong sequence homology also exists to another microbial alpha-amylase inhibitor, tendamistat (HOE 467). This paper discusses the role of a common partial sequence, -Gln-Ser-Trp-Arg-Tyr-, present in the loop of both inhibitors as the active site of microbial peptide alpha-amylase inhibitors.     

The primary structure of alpha-amylase inhibitor Z-2685 from Streptomyces parvullus FH-1641. Sequence homology between inhibitor and alpha-amylase

The native and oxidized alpha-amylase inhibitor Z-2685, isolated from the culture medium of Streptomyces parvullus FH-1641, and its overlapping cleavage products were degraded by the automatic Edman technique. Digestion was carried out with pepsin, thermolysin and trypsin. The alpha-amylase inhibitor is a polypeptide consisting of 76 amino acids with a molecular mass of 8 129 Da. With the exception of methionine and lysine, all naturally occurring amino acids are present. It is interesting that identical regions exist, in particular the sequence Trp-Arg-Tyr common to all four known microbial inhibitor sequences. We believe that the side chains of these three amino acids are important for interacting with the alpha-amylase molecule. Computer alignment enabled us to show a possible binding region in the alpha-amylase molecule which might react with the inhibitors. Furthermore, homology exists to mammalian alpha-amylases. This result is explained by the assumption that the inhibitor evolved from a duplication of the original gene of the enzyme.     

Determination of proteins in infant formula by high-performance liquid chromatography-electrospray tandem mass spectrometry

To determine the protein content of formula, gel electrophoresis was performed on the infant formula samples and the entire protein patterns were analyzed by nano-high performance liquid chromatography-electrospray tandem mass spectrometry (nano-HPLC/ESI/MS/MS). From the commercial infant formula profiled in this study, a total of 154 peptides, corresponding to 31 unique proteins were identified by nano-HPLC/ESI/MS/MS. Each of the identified peptides was reconfirmed by a strict integrated approach using tandem mass spectra. This protein profiling method using gel electrophoresis coupled with nano-HPLC/ESI/MS/MS and manual evaluation is a sensitive and accurate method for protein identification as well as a powerful tool for monitoring various types of food products.     

Determination of glycan structure from tandem mass spectra

Glycans are molecules made from simple sugars that form complex tree structures. Glycans constitute one of the most important protein modifications and identification of glycans remains a pressing problem in biology. Unfortunately, the structure of glycans is hard to predict from the genome sequence of an organism. In this paper, we consider the problem of deriving the topology of a glycan solely from tandem mass spectrometry (MS) data. We study, how to generate glycan tree candidates that sufficiently match the sample mass spectrum, avoiding the combinatorial explosion of glycan structures. Unfortunately, the resulting problem is known to be computationally hard. We present an efficient exact algorithm for this problem based on fixed-parameter algorithmics that can process a spectrum in a matter of seconds. We also report some preliminary results of our method on experimental data, combining it with a preliminary candidate evaluation scheme. We show that our approach is fast in applications, and that we can reach very well de novo identification results. Finally, we show how to count the number of glycan topologies for a fixed size or a fixed mass. We generalize this result to count the number of (labeled) trees with bounded out degree, improving on results obtained using Pólya's enumeration theorem.     

Assignment of the five disulfide bridges in an alpha-amylase inhibitor from wheat kernel by fast-atom-bombardment mass spectrometry and Edman degradation.

The assignment of the five disulfide bridges in an alpha-amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast-atom-bombardment mass spectrometry (FAB-MS) and automatic sequencing based on Edman degradation. Direct FAB-MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.     

Analysis of derivatized ceramides and neutral glycosphingolipids by high-performance tandem mass spectrometry

Microscale reduction of ceramides and neutral glycosphingolipids has been evaluated as a means of improving their analysis by fast atom bombardment mass spectrometry, alone and in combination with tandem mass spectrometry. Reduction (conversion of the amide to an amine) of native ceramides and glycosphingolipids containing one to three sugars yields derivatives that show significant signal enhancement. This sensitivity increase allows the acquisition of normal and tandem fast atom bombardment mass spectra from a submicrogram amount of sample. Concomitant permethylation is required for glycosphingolipids that contain more than three sugars. Collision induced dissociation mass spectra of protonated molecular ions, recorded on a four sector instrument, show improved fragmentation allowing the simultaneous characterization of both the ceramide and carbohydrate portions of glycosphingolipids. The reductions are carried out at the nanogram to microgram level with borane, reacting the solid sample with condensed reagent vapor. The borane reduction method has been adapted for this class of substances by adding an oxidation step in order to convert unsaturated lipids to hydroxylated derivatives by oxidation of the resulting organoborane. This approach, used in conjunction with tandem mass spectrometry, allows the determination of olefinic bond location. Labeled derivatives have been prepared by reacting the substrates with trideuterioborane and were used to ascertain the fragmentations and localize olefinic bonds. The collision induced fragmentation of reduced ceramides and neutral glycosphingolipids is only weakly affected by the presence of additional functionalities, such as methoxyl (after permethylation) and hydroxyl groups (resulting from hydroboration and oxidation), a characteristic which facilitates interpretation of the spectra of unknown compounds.     

Reduction of alpha-amylase by an inhibitor from carob

Inhibitor C, one of the inhibitory fractions of the extract of Carob fruit, reduces the amount of α-amylase in the culture medium of endosperm halves of barley seed which have been treated with gibberellic acid as compared with seed halves treated with gibberellic acid alone. A similar reduction is found in the α-amylase produced by embryo halves of barley seed which have not been treated with gibberellic acid.     

Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

Determination of amiodarone and desethylamiodarone in human plasma by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with an ion trap detector

A sensitive and specific high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) method has been developed for the simultaneous determination of amiodarone and desethylamiodarone in human plasma. After the addition of the internal standard tamoxifen, plasma samples were extracted using Oasis MCX solid-phase extraction cartridges. The compounds were separated on a 5 microm Symmetry C18 (Waters) column (150 x 3.0 mm, internal diameter) with a mobile phase of acetonitrile-0.1% forrmic acid (46:54, v/v) at a flow-rate of 0.5 ml/min. The overall extraction efficiency was more than 89% for both compounds. The assay was sensitive down to 1 microg/l for amiodarone and down to 0.5 microg/l for desethylamiodarone. Within-run accuracies for quality-control samples were between 95 and 108% of the target concentration, with coefficients of variation <8%. The proposed method enables the unambiguous identification and quantitation of amiodarone and desethylamiodarone in both clinical and forensic specimens.     

Determination of the substance P receptor antagonist CP-122, 721 in plasma by narrow-bore high-performance liquid chromatography-ionspray tandem mass spectrometry

A simple, highly sensitive and specific LC-MS-MS assay was developed for the determination of CP-122,721 (I) in rat and human plasma. I and a structural analog, CP-129,943 (II, internal standard), were extracted from plasma with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were reconstituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC column and a mobile phase of acetonitrile-20 mM ammonium acetate, pH 5 (50:50, v/v). Column effluents were monitored by ionspray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to product ion combinations of m/z 381 → 205 and 395 → 219 was used to quantitate I and II, respectively. The assay exhibited a linear dynamic range of 0.2–100 ng/ml. Absolute recoveries from plasma were above 80% for both I and II. The precision and accuracy values for the method were within ±3 and ±9%, respectively. Sample analysis times were less than 5 min from one injection to the next. The assay has proved to be applicable to the pharmacokinetic study of I in rats.     

Secondary structure of the alpha-amylase polypeptide inhibitor tendamistat from Streptomyces tendae determined in solution by 1H nuclear magnetic resonance

Complete sequence-specific 1H nuclear magnetic resonance assignments were obtained for the backbone hydrogen atoms in Tendamistat, a protein with 74 residues. From NOESY observation of 1H-1H short distance constraints, measurements of the spin-spin couplings 3JHN alpha and a qualitative identification of slowly exchanging amide protons, two antiparallel beta-sheets containing three and four strands, respectively, were identified. The peptide segments outside the beta-sheets do not form regular secondary structure. Preliminary data were obtained on the relative spatial arrangements of the two beta-sheets.     

Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry

Glycosphingolipids and gangliosides have been investigated by using fast atom bombardment high-performance tandem mass spectrometry (FABMS/MS). Homologous compounds have been investigated in order to ascertain the fragmentation. Collision-induced dissociation spectra of the molecular species in the positive ion mode mainly afford information on the ceramide constitution (aglycon as a whole, N-acyl residue, and long-chain base), whereas negative ion spectra show fragments informative of the sugar sequence and the degree of branching of the carbohydrate. In the case of gangliosides carrying a complex oligosaccharide moiety, collision spectra of fragment ions have been performed in order to gain additional structural data. The advantage of tandem mass spectrometry over conventional fast atom bombardment mass spectrometry (FABMS) consists in the fact that collision spectra of the individual components from mixtures, as usually encountered with these kinds of samples, can be recorded. Furthermore, the exclusion of most of the interfering signals from the matrix allows the identification of pertinent fragments at low mass.     

Determination of nifedipine in human plasma by flow-injection tandem mass spectrometry

For use in clinical studies, a fast and sensitive assay method was developed for the determination of nifedipine in human plasma samples. The assay method is based on tandem mass spectrometry detection (HPLC–MS–MS). The effect of flow injection as well as HPLC separation on the results of the nifedipine determination were evaluated. The limit of quantification is 0.5 ng/ml and the accuracy (as determined by spiking recovery) was found to be good.     

Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae.

The coding region for a secreted proteinaceous inhibitor of the human alpha-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. The gene is part of a 37-kilobase amplified genomic sequence found in an overproducing mutant of Streptomyces tendae. After subcloning, sequence analysis revealed an open reading frame of 312 base pairs preceded by a putative ribosome-binding site. The reading frame is 30 codons longer than necessary for the mature protein. This sequence coded for an amino-terminal extension of tendamistat and shows typical features of a signal peptide. After being cloned into Streptomyces vector plasmids and transformed to the heterologous host, Streptomyces lividans TK24, the gene was expressed, and the alpha-amylase inhibitor was correctly processed and secreted into the culture medium. The amount of secreted protein was dependent on the gene dosage and on the promoter arrangement.     

Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.