Mass Spectrometric Methodology Research Articles

Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection

Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the anti-rotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86–258) and a small peptide (324–329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf.

Characterization of C2S4·+ isomers by mass spectrometry and ab initio molecular orbital calculations

Isomeric C2S4·+ radical cations, a dimer (CS2)CS2·+ and a covalently bounded C2S4·+ species, are generated by chemical ionization (self-chemical ionization conditions) of carbon disulfide or by dissociative electron ionization of 1,3,4,6-tetrathiapentalene-2,5-dione and 1,2-dithiolo[4,3-c][1,2]dithiole-3,6-dione, respectively. These ions are structurally characterized making use of mass spectrometric methodologies such as collisional activation, neutralization–reionization and ion–molecule reactions performed in a hybrid tandem mass spectrometer of sector–quadrupole–sector configuration. These experimental results are supported by ab initio calculations of the relative energies of 19 possible isomers indicating that the most stable C2S4·+ structure corresponds to a CS2 dimer with a C2 symmetry, 52 kJ mol−1 more stable than the lowest-energy covalently bound structure consisting of a four-membered ring thioketene species.

Facile sequencing of oligosaccharides by matrix-assisted laser desorption/ionisation on a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer.

N-Linked oligosaccharide mixtures released from a number of standard glycoproteins were derivatised with 3-acetylamino-6-acetylaminoacridine (AA-Ac) using reductive amination. Analysis of these mixtures using an experimental matrix-assisted laser desorption/ionisation (MALDI) hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer provided detailed information about the mass distribution of the glycan derivatives. Collision-induced dissociation of the singly protonated [M + H](+) ions also gave rise to a number of product ions produced by the sequential cleavage of the glycosidic linkages. As fragmentation of the positively charged species occurred predominantly in one direction, i.e., from the non-reducing end of the glycan to the AA-Ac moiety, a considerable amount of information could be obtained with ease about the sequence in which the sugar residues were attached to one another. This derivatisation procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from proteins separated by gel electrophoresis.

Mass spectrometric methodology for the analysis of highly oxidized diterpenoid acids in Old Master paintings.

Diterpenoid resins from larch and pine trees and the corresponding fractions in a >100-year-old wax-resin adhesive and varnish and a 200-year-old resin/oil paint sample were analysed with by gas chromatography/mass spectrometry (GC/MS) using several off-line and on-line derivatization methods. The main resin compounds were highly oxidized abietic acids. Important products found are hydroxydehydroabietic acids (OH-DHAs), 7-oxoDHA, di-OH-DHAs and 15-OH-7-oxoDHA. The last two compounds have not been reported to occur in artworks before. Larixyl acetate, an important marker from larch resins, was found to be still present in high amounts in the adhesive. A large number of mass spectra of the different oxidation products and larixol and larixyl acetate are presented and their fragmentation behaviour under electron impact conditions is discussed. An index for the degree of oxidation (IDOX) of the abietic acids is presented as an indicator of the degree of oxidation of the matrix in which the resin is present. The IDOX was 0.10, 0.67, 0.81 and 0.76 for the fresh resins, the dark-aged adhesive, the aged varnish and the resin/oil paint, respectively (measured with pyrolysis (Py)-tetramethylammonium hydroxide (TMAH)-GC/MS). Py-TMAH-GC/MS and direct temperature-resolved mass spectrometry are reliable, valuable and fast techniques for the assessment of the presence and degree of oxidation of diterpenoid resins. Copyright 2000 John Wiley & Sons, Ltd.

Topology of the calmodulin-melittin complex

The topology of the Ca2+-calmodulin-melittin ternary complex has been investigated by a combined strategy which integrates limited proteolysis and cross-linking experiments with mass spectrometric methodologies. The rationale behind the methods is that the interface regions of two interacting proteins are accessible to the solvent in the isolated molecules, whereas they become protected following the formation of the complex. Therefore, when limited proteolysis experiments are carried out on both the isolated proteins and the complex, differential peptide maps are obtained from which the interface regions can be inferred. Alternatively, cross-linking reactions performed under strictly controlled conditions lead to the identification of spatially closed amino acid residues in the complex. Mass spectrometry can be employed in both procedures for the definition of the cleavage sites and to identify covalently linked residues.Our results show that melittin interacts with calmodulin by adopting a parallel orientation, i.e. the N and C-terminal halves of the peptide are anchored to the amino and carboxy-terminal domains of the protein, respectively. This orientation is inverted with respect to all the peptide substrates examined so far. A model of the complex was designed and refined on the basis of the experimental results, supporting the above conclusions. This finding reveals a further dimension to the already remarkable capability of calmodulin in binding different protein substrates, providing this protein with the capability of regulating an even larger number of enzymes.

Analysis of human serum albumin variants by mass spectrometric procedures

A new strategy for the structural characterisation of human albumin variants has been developed which makes extensive use of mass spectrometric methodologies. The rationale behind the method is to provide a rapid and effective screening of the entire albumin structure. The first step in this strategy consists in the attempt to determine the accurate molecular mass of the intact variant by electrospray mass spectrometry often providing a first indication on the presence of the variant. An HPLC procedure has been developed to isolate all the seven fragments generated by CNBr hydrolysis of HSA in a single chromatographic step. A rapid screening of the entire albumin structure is achieved by the ESMS analysis of the peptide fragments and the protein region(s) carrying the structural abnormality is identified by its anomalous mass value(s). Mass mapping of the corresponding CNBr peptide, either by Fast Atom Bombardment Mass Spectrometry (FABMS) or by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDIMS), leads to the definition of the site and the nature of the variation. This combined strategy was applied to the structural characterisation of three HSA genetic variants and provided to be an effective procedure for the rapid assessment of their structural modifications showing considerable advantages over the classical approach.

Sensitive detection and structural characterization of trimethyl(p-aminophenyl)-ammonium-derivatized oligosaccharides by electrospray ionization-mass spectrometry and tandem mass spectrometry.

Mass spectrometric methodologies which use electrospray ionization (ESI) for oligosaccharide structural determination and which are linked to classical procedures, such as reductive amination, have been established. Several derivatives of a model oligosaccharide were prepared and their ionization efficiencies in electrospray ionization (ESI) and fast-atom bombardment (FAB) mass spectrometry were investigated. The trimethyl-(p-aminophenyl)ammonium (TMAPA) derivatives showed extremely high sensitivity in positive-ion ESI-MS and gave Y- and Z-series sequence ions by ESI tandem mass spectromety (MS/MS). This study suggests that TMAPA-derivatized oligosaccharides used in ESI-MS and ESI-MS/MS would be effective for structural characterization of oligosaccharides that are available only in limited quantities.

Nonreducing two-dimensional polyacrylamide gel electrophoretic analysis of human colonic proteins.

Immunochemical detection of proteins with antigenic determinants that are dependent on the native spatial conformation of the protein can often pose problems with conventional two-dimensional polyacrylamide gel electrophoresis (2-DE). For example, many antigenic determinants are readily destroyed by reducing agents and/or urea, reagents which are critical components of many of the conventional isoelectric focusing and immobilized-pH-gradient (IPG) protocols used in the first electrophoretic dimension. Here we describe the use of commercially available precast 2-DE gels for performing nonreducing/non-urea 2-DE of proteins extracted from the human colon cancer cell line LIM 1215 with 0.3% Triton X-100 that permit the identification of antigens with conformational determinants by immunoblot analysis. Previous, related studies demonstrated the usefulness of peptide-mass fingerprinting for identifying 2-DE resolved proteins. Here we show how partial protein sequence data obtained by rapid peptide mapping, using capillary column liquid chromatography directly coupled with electrospray ionization tandem mass spectrometric methodologies, enhances the usefulness of this approach for identifying incompletely resolved proteins. The nonreducing 2-DE gel images reported in this study, along with our master 2-DE gel protein database for both normal human colonic crypts and several colon-cancer-derived cell lines, and information regarding microtechniques employed in this laboratory for obtaining structural data on 2-DE resolved proteins can be accessed over the Internet using World Wide Web (URL address: http:@www.ludwig.edu.au).

Recent Advances in Mass Spectrometry.

Amongst other examples of modern technology, mass spectrometry (MS) has made extraordinary progress in the last decade in terms of sensitivity, precision and the possible range of mass measurement, progress which could not be foreseen ten years ago. Starting in 1970, a method after the method of ionization was rapidly developed in tandem with dramatic improvements and innovations in MS equipment, as a result of which the range of applications of mass spectrometry has expanded exponentially, allowing MS to assume very important new roles in various fields of the life sciences, including medicine, pharmacy and environmental chemistry. One of the most outstanding developments in MS in recent years is the ability to measure non-volatile substances such as protein, nucleic acid, glycolipids, polysaccharide, and high polarity macromolecules. The most important contribution in this development come mostly from fast atom bombardment (FAB) ionization, the latest state-of-the-art ionization methods such as electrospray (ES) ionization or ionspray (IS) ionization, new range of mass spectrometric methodology, tandem mass spectrometry (MS/MS) and time of flight mass spectrometry (TOF-MS). This review centers on an outline of the mass spectrometry of non-volatile, high polarity macromolecules, concentrating on FAB, ES, IS, MS/MS and TOF-MS.

A structural study on elcatonin, a novel synthetic analogue of eel calcitonin, by fast atom bombardment and tandem mass spectrometry.

Elcatonin (ELC), a synthetic analogue of eel calcitonin, successfully used in the treatment of diseases characterized by an increase of osteoclastic activity, has been fully sequenced by combined enzymatic hydrolysis and fast atom bombardment (FAB) mass spectrometric methodology. The FAB mass spectrometric analysis on the entire molecule gave only the cluster corresponding to the molecular ion. Digestion with trypsin afforded four oligopeptides corresponding to fragments 1-11, 12-18, 19-24 and 25-32. B/E daughter ion analysis performed in turn on each protonated molecule ion in the tryptic mixture allowed complete sequencing of fragments 1-11 and 12-18, while in fragments 19-24 and 25-32 the portions 23-24 and 25-26 remained respectively unclarified. Investigation on the single oligopeptide isolated by preparative high-performance liquid chromatography and chymotryptic digestion of the molecule failed to provide any new information. One step of Edman degradation on tryptic digest permitted attribution of the 25-26 sequence to Thr-Asp. The connectivity between tryptic ELC fragments and the 23-24 sequence were proved by cleavage with V8 protease, which gave the oligopeptides 1-15 and 16-32. These were exhaustively analysed by FAB mass spectrometry.

The correct molecular weight of myoglobin, a common calibrant for mass spectrometry.

Myoglobins from horse heart muscle, horse skeletal muscle and sperm whale are widely used as calibration standards or test compounds for various mass spectrometric methodologies. In all such cases reported in the literature, a molecular weight value is used (16,950.5 and 17,199, respectively) which is based on the assumption that amino acid 122 in this 153 amino-acid-long protein is asparagine, overlooking a published suggestion that it is aspartic acid instead. Since the mass assignment accuracy for matrix-assisted laser desorption mass spectrometry is reported to be +/- 0.01% and for electrospray ionization +/- 0.0025%, and error of one mass unit in approximately 17,000 would be significant. The mass-to-charge ratio of ions of the tryptic peptide encompassing amino acid 122 derived from commercially available horse heart and horse skeletal myoglobins, the apomyoglobin of the latter, and the tryptic and chymotryptic peptide of sperm whale myoglobin proved that in both proteins amino acid 122 is indeed aspartic acid, rather than asparagine. This finding was further confirmed by the collision-induced dissociation spectra of the [M + H]+ ions of the tryptic peptides from the horse myoglobins and the chymotriptic peptide from sperm whale myoglobin. Thus, the correct molecular weight of horse myoglobin is 16,951.49 and that of the sperm whale protein is 17,199.91.

Minireview quantitative analytical mass spectrometry of endogenous neuropeptides in human pituitaries

This manuscript reviews state-of-the-art mass spectrometric (MS) methodology for the qualitative (amino acid sequence determination) and quantitative analysis of opioid neuropeptides in human pituitary tissue (1–7). Those analytical data are required for the elucidation of the basic molecular mechanisms involved in tumor formation (1) and to test the hypothesis that metabolic defects in neuropeptidergic system processing is a contributing factor to human anterior pituitary tumor formation. Several different neuropeptide products that derive metabolically from the proenkephalin A and proopiomelanocortin (POMC) precursors have been analyzed separately (4, 6–8) and together (5) in human pituitaries, including post-mortem controls and post-surgical tumors. The quantification, with optimal molecular specificity, of a peptide in a tissue is an important measurement because the amount of an endogenous peptide reflects the ratio of its synthesis to its degradation and thus, any defects in those processes may be reflected in the amount of a peptide.

Fast atom bombardment mass spectrometric strategies for characterizing carbohydrate-containing biopolymers.

Fast atom bombardment mass spectrometric methodologies for carbohydrate structure determination are intimately linked to classical procedures such as derivatization and hydrolysis. This paper reviews the current status of carbohydrate fast atom bombardment mass spectrometry and reports on two new protocols which we are using to optimize sensitivity and fragmentation.

Development of capillary gas chromatographic—mass spectrometric methodology for the simultaneous determination of ibuprofen and [ar- 2H 4]ibuprofen in serum: demonstration of kinetic equivalence in the beagle

A capillary gas chromatographic—mass spectrometric method for the determination of ibuprofen and tetra-deuterated ibuprofen in serum is described. Ibuprofen, [ar- 2H 4]-ibuprofen and the internal standard, [ar- 2H 4,3,3,3- 2H 3]ibuprofen, are extracted (after acidification) from serum onto a cross-linked styrene divinyl benzene resin by an automated sample processor. After elution and evaporation of the organic phase, samples are reconstituted with solvent and analyzed without derivatization by capillary gas chromatography—mass spectrometry. This methodology was used to evaluate possible kinetic isotope effects after the coadministration of an equimolar mixture of ibuprofen and the deuterium-labeled covariant in the beagle. No significant differences in absorption or elimination were observed.

Whole body amino acid pools and turnover rates determined by a stable isotope gas chromatographic mass spectrometric methodology.

Whole body amino acid pools and turnover rates determined by a stable isotope gas chromatographic mass spectrometric methodology.