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ABSTRACT: Most characteristics of matrix-assisted laser desorption/ionization (MALDI) are ideal for the analysis of biomolecules. New preparation techniques have dramatically increased mass accuracy and resolution, making MALDI a high-performance mass spectrometric technique for peptide mass analysis. Attempts to obtain amino acid sequence information by MALDI have been partially successful. The technique has been put to novel uses in protein primary structure characterization.
Current Opinion in Biotechnology 03/1996; 7(1):11-9. · 7.71 Impact Factor
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ABSTRACT: The possibility of structural elucidation of carbohydrates and oligonucleotides by matrix-assisted laser desorption/ionization followed by post-source decay (MALDI-PSD) is investigated. Spectra of both classes of compounds exhibit the better signal-to-noise ratios for the intact species in the negative-ion mode, but the most informative spectra for structural elucidation by PSD are obtained in the positive-ion mode. A novel fragmentation mode is demonstrated on a model sialylated complex type N-linked oligosaccharide. Prompt fragmentation in the positive-ion mode can remove the sialic acids at high laser irradiance if a suitable matrix is used. The remaining sugar backbone is then characterized by post-source decay. A modified oligonucleotide is analyzed with 3-hydroxypicolinic acid as the matrix and ammonium tartrate to displace sodium. Enough structural information is obtained to verify the modification.
Rapid Communications in Mass Spectrometry 02/1996; 10(1):100-3. · 2.79 Impact Factor
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ABSTRACT: The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI), especially at the two-disulphide stage, has been dissected by replacing one or two particular cysteine residues by serine. This restricts which disulphide species are possible, and the observed kinetics of disulphide-coupled folding reveal the roles of the remaining species. The results obtained confirm the kinetic roles in the original BPTI pathway of the two specific two-disulphide intermediates with non-native second disulphide bonds, (30-51, 5-14) and (30-51, 5-38). Moreover, the rates of folding through each of these intermediates are shown to account quantitatively for the rate of folding of the normal protein; therefore, essentially all the molecules refold through these two particular intermediates. They are amongst the most productive on the folding pathway, and their roles are readily explicable on the basis of their conformations.
Journal of Molecular Biology 07/1995; 249(2):463-77. · 4.00 Impact Factor
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ABSTRACT: When expressed in E. coli, skeletal muscle alpha-tropomyosin has an unacetylated N-terminus. Unacetylated alpha-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant alpha-tropomyosin, rabbit skeletal muscle alpha-tropomyosin (alpha-tropomyosin BV) has been expressed in baculovirus-infected insect cells. alpha-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of alpha-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from alpha-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant alpha-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectrometry indicated a single phosphorylation not only in alpha- but also beta-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of alpha-tropomyosin.
Journal of Muscle Research and Cell Motility 05/1995; 16(2):103-10. · 1.98 Impact Factor
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ABSTRACT: [M + H + 16]+ ions were observed in the electrospray ionization mass spectra of several synthetic and naturally occurring peptides. Initial results have shown that the appearance of the modification is dependent on the field strength at the electrospray needle and the flow rate of the solution passing through the capillary. Mass spectrometric experiments on peptides showing the + 16 Da modification attributed the change to the selective oxidation of either a methionyl, tryptophanyl, or tyrosyl residue present in the peptide. These results were further confirmed by tandem mass spectrometry experiments on peptides with a methionyl residue at either the N- or C-terminus, or within the peptide chain. This effect can occur under normal operating conditions and therefore care must be taken in the analysis of samples containing these oxidizable residues. Conversely, the selectivity of the oxidative process may be used to enhance the information obtained from the mass spectrometric analysis. For example, we show results for the analysis of a tryptic digest of the protein myoglobin where the occurrence of the [M + H + 16]+ ion is used, along with the molecular weight data, to correctly identify the trypic fragment.
Rapid Communications in Mass Spectrometry 09/1993; 7(8):738-43. · 2.79 Impact Factor
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ABSTRACT: Cytochrome c oxidase contains a copper center, CuA, which is involved in electron transfer from cytochrome c to the oxygen-reducing active site. This center is distinct from types 1, 2, and 3 copper sites and related only to a purple copper center in nitrous oxide reductase. At present it is not clear whether this site is mononuclear or is comprised of two copper atoms in a mixed valence (Cu(I)-Cu(II)) configuration. Here we use a model of CuA, engineered into a structurally related but initially copperless protein, to study the structure of this copper center. The results from biochemical analysis, site-directed mutagenesis, and electrospray mass spectrometry support the binuclear model. Two cysteines, two histidines, and one methionine are the major ligands of two coppers. Substitution of these residues results in either a complete loss of color or dramatic changes in the absorbance spectrum. In contrast, substitution of the invariant glutamate residue, which is located between the copper-binding cysteines, leads to a minor perturbation of the optical spectrum.
Journal of Biological Chemistry 09/1993; 268(22):16781-7. · 4.77 Impact Factor
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ABSTRACT: The parameters for recovery of sample from the nitrocellulose support used in plasma desorption are optimized. The losses in washing, in situ reactions, and extraction procedures are quantitatively evaluated. At least 80% of the sample can be effectively extracted or transferred to a polyvinyl difluoride membrane with 2-propanol-water mixture in the range 1:1-2:3 v/v. Quantitative losses of insulin during washing procedures vary from 0 to 50% depending on the washing procedure used. The losses in in situ reactions are negligible. Optimization of the procedures allows several successive procedures to be carried out after adsorption of 1 nmol of insulin on the nitrocellulose support. These include in situ reaction, extraction of A and B chains followed by S-alkylation, chain separation by high-performance liquid chromatography, mass spectrometric analysis of the separated chains, and finally automatic sequence after transfer to the sequenator.
Biological Mass Spectrometry 02/1993; 22(1):77-83.
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ABSTRACT: The complete structure of protein isolated from endocuticle of sexually mature locusts, Locusta migratoria, has been determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The protein is extensively post-translationally modified. The N-terminal is 5-oxoproline (pyroglutamic acid) and the C-terminal proline residue is amidated. Furthermore, the protein is glycosylated by a single N-acetyl-galactosamine residue at one, two or three threonines. The N-terminal sequence was obtained by analysing the N-acetylated N,O-permethylated derivative using plasma desorption mass spectrometry. The position and type of carbohydrate were determined by combining an HPLC-based carbohydrate analysis with the peak pattern of the phenylthiohydantoin derivative in automatic sequencing and with mass information on peptides. The protein has pronounced similarity to cuticular proteins from larvae of diptera and lepidoptera, but only slight resemblance to the previously sequenced locust exocuticular proteins. This indicates a similarity between soft larval cuticles and locust endocuticle, a similarity which may extend to their mechanical properties.
European Journal of Biochemistry 02/1991; 195(2):495-504. · 3.58 Impact Factor
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ABSTRACT: The plasma desorption mass spectra of N-acetylated-N,O-permethylated peptides contain sufficient fragment ions to allow partial or complete sequence determination. By optimization of the derivatization procedure and an inclusion of a purification step by high-performance liquid chromatrography (HPLC) overall sensitivities on the high picomole level are obtained. By using limiting derivatization conditions the fully derivatized peptide is easily selected from the HPLC separation. Mainly N-terminal sequence ions are observed, facilitating sequence determination of naturally N-blocked peptides. Complete sequence determination of naturally formylated gramicidin A containing 15 amino acid residues and the naturally N-acetylated N-terminal heptapeptide from an acyl-CoA binding protein is demonstrated. The sequence of the first six N-terminal residues was obtained by derivatization of the A-chain of insulin.
Biomedical & environmental mass spectrometry 11/1990; 19(10):589-96.
