Plasma desorption mass spectrometry as a tool in characterization of abnormal proteins. Application to variant human hemoglobins.

We here report the application of plasma desorption mass spectrometry in combination with reversed-phase high-performance liquid chromatography and automatic Edman sequencing for the characterization of hemoglobin variants. By use of plasma desorption mass spectrometry to obtain molecular weight information of purified globin peptides it is possible to minimize the number of candidate positions for substitutions allowing an optimal use of automatic Edman degradation. Each variant can be characterized by using less than 200 micrograms of hemoglobin, corresponding to approximately 2 microliters of blood, as starting material. The outlined approach is considered to be very well suited for routine analysis of hemoglobins and other protein variants, natural as well as recombinant.     

Disulfide structure of alfimeprase: a recombinant analog of fibrolase

The disulfide structure of alfimeprase, a recombinant analog of fibrolase, was experimentally determined by a combination of peptide mapping, Edman degradation, and mass spectrometry. The three disulfide bonds were determined to be Cys-116/196, Cys-156 /180, and Cys-158/163 with the residue number system of alfimeprase.     

Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis.

Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1h post challenge. Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2kDa, was determined by MALDI-TOF mass spectrometry. N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K. A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70 degrees C. Although the tick defensin alone was not immediately effective against B. burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B. burgdorferi within 1h.     

Primary structure of three cationic peptides from porcine neutrophils Sequence determination by the combined usage of electrospray ionization mass spectrometry and Edman degradation

The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.     

Amino acid sequence determination of a protein purified from the shell of the shrimp,Pandalus borealis

One of the urea-extractable proteins in the shell of the shrimp Pandalus borealis has been purified, and the complete amino acid sequence has been determined by the combined use of mass spectrometry and Edman degradation of the intact protein as well as of enzymatically derived peptides.Characteristic features of the sequence are discussed and compared to sequences from insect cuticular proteins and other structural proteins.     

Acyl-CoA-binding protein in the rat. Purification, binding characteristics, tissue concentrations and amino acid sequence.

Acyl-CoA-binding protein (ACBP) was purified from rat liver. The Mr was determined as 9932 +/- 10 by mass spectrometry and calculated as 9937.8 from the sequence. The protein binds acyl-CoA esters (C8-C16) with high affinity, but was unable to bind fatty acids. ACBP was found mainly (86%) in the soluble fraction, and the concentration was highest in liver, 5-6 micrograms/mg of soluble protein. The complete primary structure was determined by a combination of gas-phase Edman degradations and mass spectrometry. Extensive use of 252Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides. Comparison with the previously determined sequence of bovine acyl-CoA-binding protein revealed a very strong sequence similarity (83%), and all of the differences could be accounted for by single base changes.     

Sequence determination of three cuticular proteins and isoforms from the migratory locust,Locusta migratoria,using a combination of Edman degradation and mass spectrometric techniques

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.     

Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity.

A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host-pathogen interactions. The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection. The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration. Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry. They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides. Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria. Anomala defensin B was active at high concentration of 40 microM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A. cuprea larvae.     

Sequence determination of three cuticular proteins and isoforms from the migratory locust,Locusta migratoria,using a combination of Edman degradation and mass spectrometric techniques

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.     

Amino acid sequence of acyl-CoA-binding protein from cow liver.

Acyl-CoA-binding protein from bovine liver was purified with the use of reverse-phase h.p.l.c. in the final step. The complete amino acid sequence was determined by using a combination of gas-phase Edman degradation and electron-impact and fast-atom-bombardment mass spectrometry. The sequence was confirmed by determination of the Mr by plasma-desorption time-of-flight mass spectrometry.     

Purification, partial sequencing and characterization of an insect membrane dipeptidyl aminopeptidase that degrades the insect neuropeptide proctolin.

Two proctolin-binding proteins solubilized from 1600 cockroach hindgut membranes were purified 1000-fold using five chromatography steps. Twenty-five micrograms of protein were recovered from the final size-exclusion chromatography as a single peak eluting at 74 kDa, whereas two major bands at 80 and 76 kDa were identified after silver staining of electrophoresis gels. The fragments, sequenced by tandem mass spectrometry and the Edman method, revealed a high homology with rat liver dipeptidyl aminopeptidase (DPP) III and a significant homology between the cockroach-purified proteins. From analysis of the Drosophila genome sequence database, it was possible to identify a putative DPP sharing high homology with the sequences obtained from the cockroach purified proteins and with the rat DPP III. Anti-(rat liver DPP III) Ig reacted specifically with both cockroach-purified proteins in Western blot analysis. The purified proteins removed the N-terminal dipeptide from the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) with a Km value of 3.8 +/- 1.1 microM. The specific DPP III inhibitor tynorphin prevented the degradation of proctolin by the purified insect DPP (IC50 = 0.68 microM). These results provide strong evidence that the cockroach-purified proteins represent an insect membrane DPP, presumably present in Drosophila, and that it is closely related to vertebrate DPP III.     

Identification of multiple peptides homologous to cockroach and cricket allatostatins in the stick insect Carausius morosus

Eighteen peptides were isolated from brain extracts of the stick insect Carausius morosus. The peptides were purified in four steps by high-performance liquid chromatography, monitored by their ability to inhibit juvenile hormone biosynthesis by corpora allata of the cricket Gryllus bimaculatus in vitro, and chemically characterised by Edman degradation and mass spectrometry. We obtained complete primary-structure information for nine peptides, four of which belong to the peptide family characterised by a common C-terminal pentapeptide sequence -YXFGLamide. The remaining five belong to the W(2)W(9)amide peptide family, nonapeptides characterised by having the amino acid tryptophan in positions 2 and 9. The amino-acid sequence of two other peptides could not be completely resolved by means of Edman degradation; however, these peptides could be allocated to the -YXFGLamide and the W(2)W(9)amide family, respectively, by comparison of retention times, co-elution and mass spectrometry. Both classes of neuropeptides strongly inhibit juvenile hormone biosynthesis in crickets but show no inhibiting effect on the corpora allata of the stick insect.     

Mass spectrometry of natural and recombinant proteins and glycoproteins

Most modern protein sequence analysis is carried out using classical, wet-chemical Edman degradation technology. However, an increasing number of studies on both natural and recombinant genetically engineered proteins demands the use of new technologies capable of assigning structural features such as glycosylation, which cannot be assigned by Edman sequence analysis. The most important alternative and complementary procedure at present is the use of high-mass mass spectrometry. This brief article introduces some of the principles and applications of the technique. Protein research laboratories, both academic and industrial will make increasing use of these techniques to complement classical gas phase sequencing, and to identify post-translational modifications including glycosylation, phosphorylation, SS bridge assignment and processing events, including the formation of ‘ragged ends’.     

A biologically active hydrophobic T-1-conotoxin from the venom of Conus spurius

A major, very hydrophobic peptide, sr5a, was purified from the venom duct of Conus spurius specimens collected in the Yucatan Channel, Mexico. Its amino acid sequence (IINWCCLIFYQCC; calculated monoisotopic mass assuming two disulfide bridges 1616.68 Da) was determined by automatic Edman degradation after reduction and alkylation, and confirmed by mass spectrometry (ESI monoisotopic mass, 1616.60; MALDI monoisotopic mass 1616.42 Da). The primary structure of sr5a showed the pattern that characterizes the family of the T-1-conotoxins, which belong to the T-superfamily of conotoxins. The disulfide bonds were determined by partial reduction and alkylation with N-ethylmaleimide, followed by total reduction and alkylation with 4-vinylpyridine, and automatic Edman sequencing. The connectivity of the Cys residues (I-III, II-IV) is the same as that found in the T-1-conotoxin family. When injected intracranially (2.0 nmol) into mice, peptide sr5a caused depressed behavioral activity.     

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.     

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.     

A recombinant functional variant of the olive pollen allergen Ole e 10 expressed in baculovirus system

Pollens have been reported as important sources of antigens causing type-I allergy and, among them, olive pollen has high clinical relevance in Mediterranean countries. The most recently described olive allergen, Ole e 10, is involved in cross-reactivity phenomena and related to asthma induction in allergic patients. These immunologic features make this allergen a good candidate to be included in diagnosis and therapy of protocols of allergic diseases. Since the availability of Ole e 10 from the olive pollen is limited, the allergen has been efficiently expressed in the baculovirus/insect cell system. The Ole e 10-cDNA inserted into the transfer vector pBacPAK8 allowed the expression of the recombinant protein in cultured Sf21 cells. Recombinant Ole e 10 (rOle e 10) was purified from the culture after dialysis and three chromatographic steps. Mass spectrometry, Edman degradation, IgE- and IgG-binding analyses were employed to characterize the recombinant allergen, which showed molecular and immunological equivalence with the natural protein. Affinity gel electrophoresis in presence of laminarin (1,3-beta-glucan) revealed that rOle e 10 retains identical carbohydrate-binding capacity than the natural allergen. In conclusion, the recombinant expression of Ole e 10 in baculovirus/insect cell system produces a homogeneous and biologically active allergen that could be useful for clinical and scientific purposes.     

The amino acid sequence of a major protein component in the light harvesting complex of the green photosynthetic bacterium Chlorobium limicola f. thiosulfatophilum

A 7.5-kDa protein has been isolated from chlorosomes of Chlorobium limicola f. thiosulfatophilum and the complete primary structure determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The 74-residue protein shows great homology to a similar protein of unknown function which has been isolated from Pelodictyon luteolum but otherwise no significant homology to other proteins can be found. The possible role of the protein in the structure and function of the chlorosome is discussed.     

Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure

A cationic peptide, designated tachyplesin, was isolated from acid extracts of horseshoe crab (Tachypleus tridentatus) hemocyte debris. It consists of 17 residues and the structure determined by Edman degradation is: (formula; see text) The carboxyl-terminal end of this peptide was identified as arginine alpha-amide, and the whole sequence including the alpha-amide was also confirmed by fast atom bombardment mass spectrometry, indicating a mass value of 2263. Tachyplesin inhibits growth of both Gram-negative and -positive bacteria at low concentrations and formed a complex with bacterial lipopolysaccharide. Tachyplesin seems likely to act as antimicrobial peptide for self-defense in the horseshoe crab against invading microorganisms.     

Sequence and expression of Thai Rosewood beta-glucosidase/beta-fucosidase, a family 1 glycosyl hydrolase glycoprotein

Dalcochinin-8'-O-beta-glucoside beta-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both beta-glucosides and beta-fucosides. To determine its structure and evolutionary lineage, the sequence of the enzyme was determined by peptide sequencing followed by PCR cloning. The cDNA included a reading frame coding for 547 amino acids including a 23 amino acid propeptide and a 524 amino acid mature protein. The sequences determined at peptide level were found in the cDNA sequence, indicating the sequence obtained was indeed the dalcochinase enzyme. The mature enzyme is 60% identical to the cyanogenic beta-glucosidase from white clover glycosyl hydrolase family 1, for which an X-ray crystal structure has been solved. Based on this homology, residues which may contribute to the different substrate specificities of the two enzymes were identified. Eight putative glycosylation sites were identified, and one was confirmed to be glycosylated by Edman degradation and mass spectrometry. The protein was expressed as a prepro-alpha-mating factor fusion in Pichia pastoris, and the activity of the secreted enzyme was characterized. The recombinant enzyme and the enzyme purified from seeds showed the same K(m) for pNP-glucoside and pNP-fucoside, had the same ratio of V(max) for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-beta-glucoside.