Crystallization and characterization of an inflammatory lectin purified from the seeds of Dioclea wilsonii

DwL, a lectin extracted from the seeds of Dioclea wilsonii, is a metalloprotein with strong agglutinating activity against rabbit and ABO erythrocytes, inhibited by glucose and mannose. DwL was purified by affinity chromatography on a Sephadex G-50 column and ion exchange chromatography on a HiTrap SP XL column. SDS-PAGE revealed three electrophoretic bands corresponding to the α (25,634 ± 2 Da), β (12,873 ± 2 Da) and γ (12,779 ± 2 Da) chains. Protein sequencing was done by Tandem Mass Spectrometry. The primary sequence featured 237 amino acids and was highly homologous to other reported Diocleinae lectins. A complete X-ray dataset was collected at 2.0 ? for X-Man-complexed DWL crystals produced by the vapor diffusion method. The crystals were orthorhombic and belonged to the space group I222, with the unit-cell parameters a = 59.6, b = 67.9 and c = 109.0 ?. DWL differed in potency from other ConA-like lectins and was found to induce neutrophil migration in rats, making it particularly useful in structural/functional studies of this class of proteins.     

Structural characterisation of flavonoids and flavonoid-O-glycosides extracted from Genista tenera by fast-atom bombardment tandem mass spectrometry

Genista tenera is a plant native to the Madeira Island (Portugal). From the ethanol extract of its powdered aerial parts, two flavones, three isoflavones and one 7-O-glucosyl isoflavone were isolated. A mass spectrometric study of these compounds was performed using liquid secondary ion mass spectrometry (LSIMS) in combination with high-energy collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS). Characteristic fragmentation patterns were observed in all the investigated compounds; the loss of small neutral species from the protonated molecules was useful for identifying the presence of specific functional groups in the A- and B-rings. In order to help to establish the proposed structures, NMR and UV studies were also performed.     

Structural determination of monoacylglycerols extracted from marine sponge by fast atom bombardment tandem mass spectrometry

Five new monoacylglycerols (MAGs) were isolated from the marine sponge Stelletta sp. by reversed-phase high-performance liquid chromatography and analyzed by positive ion fast atom bombardment mass spectrometry (FAB-MS). FAB mass spectra of these compounds produced abundant sodium-adducted molecules [M+Na]+ from a mixture of 3-nitrobenzyl alcohol and sodium iodide. The structural elucidation of these sponge MAGs was carried out by FAB tandem mass spectrometry (MS/MS). To find diagnostic ions for the characterization of the MAGs, authentic MAGs were initially analyzed by collision-induced dissociation (CID) MS/MS. The CID MS/MS of [M+Na]+ precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge-remote fragmentation (CRF) provided important information for the characterization of acyl chains substituted at the glycerol backbone, and product ions at m/z 84, 97, 113 and 139 were diagnostic for the sodiated glycerol backbone. On the basis of these fragmentation patterns, the structures of five MAGs extracted from marine sponge were elucidated. In addition, high-resolution mass measurement was performed to obtain the elemental compositions of the MAGs.     

Structural determination of lysophosphatidylcholines extracted from marine sponges by fast atom bombardment tandem mass spectrometry

A series of lysophosphatidylcholines were isolated from the marine sponge Spirastrella abata by reversed-phase high performance liquid chromatography (HPLC) and analyzed by fast atom bombardment mass spectrometry (FAB-MS). Their structural elucidation was carried out with fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The collision-induced dissociation (CID) of protonated and sodiated molecular ions produced diverse product ions via a series of dissociative processes. Because of the positive charge of the amine group at the end of the molecules, charge-remote fragmentation patterns of specific ions, [M + H](+) or [M + Na](+), were very helpful for the identification of product ions which are characteristic for choline and long hydrocarbon chains substituted at the glycerol back bone. Moreover, the CID-MS/MS spectra of sodium adducted molecular ions for lysophosphatidylcholines yielded common characteristic fragment ions for the choline moiety and characteristic ions [M + Na-103](+), [M + Na-85](+) and [M + Na-59](+) in the higher mass region.     

'Top down' protein characterization via tandem mass spectrometry

Technological and scientific advances over the past decade have enabled protein identification and characterization strategies to be developed that are based on subjecting intact protein ions and large protein fragments directly to tandem mass spectrometry. These approaches are referred to collectively as 'top down' to contrast them with 'bottom up' approaches whereby protein identification is based on mass spectrometric analysis of peptides derived from proteolytic digestion, usually with trypsin. A key step in enabling top down approaches has been the ability to assign tandem mass spectrometer product ion identities, which can be done either via high resolving power or through product ion charge state manipulation. The ability to determine product ion charge states has permitted studies of the reactions, including dissociation, ion-molecule reactions, ion-electron reactions and ion-ion reactions of high-mass, multiply charged protein ions. Electrospray ionization combined with high magnetic field strength Fourier transform ion cyclotron resonance has proven to be particularly powerful for detailed protein characterization owing to its high mass resolution and mass accuracy and its ability to effect electron capture-induced dissociation. Other types of tandem mass spectrometers are also beginning to find increasing use in top down protein identification/characterization studies. Charge state manipulation via ion-ion reactions in electrodynamic ion traps, for example, enables top down strategies to be considered using instruments with relatively modest mass resolution capabilities. Precursor ion charge state manipulation techniques have also recently been demonstrated to be capable of concentrating and charge-state purifying proteins in the gas phase. Advances in technologies applied to the structural analysis of whole protein ions and in understanding their reactions, such as those described here, are providing new options for the study of complex protein mixtures.     

High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery

Alterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. High-throughput glycan profiling can now be achieved with the recent developments in MS-based techniques. To enable identification and rapid monitoring of glycosylation changes in serum proteins, we developed a semi-automated high-throughput glycoprotein biomarker discovery platform termed lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS) which includes (i) effective single-step serum glycoprotein isolation using a panel of 20 individual lectin-coated magnetic beads in microplate format, (ii) on-bead trypsin digestion, and (iii) nanoLC-MS/MS with lectin exclusion list. With use of appropriate sequence databases, LeMBA-MS can detect glycosylation changes regardless of the species. By spiking known amounts of titrated ovalbumin to a serum sample, we report nanomolar sensitivity, and linearity of response of LeMBA-MS using concanavalin A-coupled beads. Neuraminidase treatment led to reduction of binding to sialic acid-binding lectins. Interestingly, we found that desialylation caused increased binding of haptoglobin and hemopexin to mannose-specific lectins, pointing to the importance of identifying a signature of lectin-binding. High-throughput LeMBA-MS to generate glycosylation signatures will facilitate glycobiomarker discovery. LeMBA can be coupled to down-stream detection platforms for validation, making it a truly versatile platform.     

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.     

Structural characterization of reactive dyes using liquid secondary ion mass spectrometry/tandem mass spectrometry

Reactive Blue 19 (RB 19), its reactive form (RB 19-VS) and its hydrolyzed form (RB 19-OH) were examined using liquid secondary ion mass spectrometry/tandem mass spectrometry (LSIMS/MS/MS) in the negative-ion mode under low-energy collision conditions (240–300 eV). Structurally characteristic fragment ions were obtained, none of which has been previously reported for these reactive dyes. Among the ions obtained were SO₃^? ions, ions due to central amino cleavage and reactive group cleavage, and ions due to loss of SO₃ and SO₂. Possible pathways for the formation of product ions are proposed. The structural information obtained should help to characterize and identify reactive dyes better.     

Structural characterization of mono- and bisphosphonium salts by fast atom bombardment mass spectrometry and tandem mass spectrometry

Positive-ion fast atom bombardment (FAB) mass spectra are reported for a representative series of mono- and bisphosphonium halides derived from triphenylphosphine. The mass spectra of the monoalkyltriphenylphosphonium salts typically contain abundant intact cations that can be used to establish the cationic relative molecular mass and diagnostic fragment ions that allow the characterization of structural subgroups. Depending on the functional group substitution on the alkyl group, additional fragment ions are observed which are formed by loss of small neutral molecules from the intact cation and that can be used for the differentiation of isomeric phosphonium salts. Molecular dication are typically observed in the FAB mass spectra of the bisphosphonium salts when they are analysed in 3-nitrobenzyl alcohol. In addition, production of singly charged ions by clustering with a counter ion, decomposition involving removal of one of the charge centres and one-electron reduction are generally observed. Structurally diagnostic fragments are also obtained. The fragmentation pathways of the ions derived from the phosphonium salts were elucidated by precursor ion and product ion tandem mass spectrometric experiments. For the phosphonium salts containing a long-chain hydrocarbon alkyl group, high-energy collision-induced decomposition of the intact cation is needed to obtain unambiguous structural information.     

Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

Bovine insulin was glycated under hyperglycemic reducing conditions and in nonreducing conditions. Purification through HPLC allowed isolating glycated forms of insulin and a novel triglycated form (6224.5 Da) was purified. Endoproteinase Glu-C digestion combined with mass spectrometry (MALDI-TOF/TOF) allowed determining the exact location of the glycation sites in each of the isolated glycated insulins. For the first time, a triglycated form of insulin was isolated and characterized accordingly to its glycation sites. These glucose binding sites were identified as the N-terminals of both chains (Gly1 and Phe1) and residue Lys29 of B-chain. Moreover, in diglycated insulin we found the coexistence of one specie glycated at the N-terminals of both chains (Gly1 and Phe1) and another specie containing the two glucitol adducts in B-chain (Phe1 and Lys29). Also, in monoglycated insulin generated in reducing and nonreducing conditions, one specie glycated at Phe1 and another specie glycated at Lys29, both B-chain residues coexist.     

Electrospray mass spectrometry and tandem mass spectrometry of the natural mixture of cyclic peptides from linseed

The cyclic peptides from linseed are composed exclusively of the hydrophobic amino acids: Phe, Leu, Ile, Val, Met, Pro, and Trp. Because these compounds does not contain functional groups which undergo easily protonation or deprotonation. their ionization in solvents used usually for peptide analysis is not efficient. A rapid and sensitive procedure for detection and structure elucidation of the cyclic peptides based on ionization with Na+ and NH4+ ions. A cationisation of methionine containing peptides with methyl iodide has been also described. The extract of seeds of Linum utitatissimum was analyzed directly by ESI-MS and neutral loss ESI-MS/MS technique. The analysis confirms the presence of cyclolinopeptides reported previously: CLA (c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), and CLB (c(Pro-Pro-Phe-Phe-Val-Ile-Met-Ile-Leu)). Cyclolinopeptides CLC, CLD, CLE, and CLG, which contain methionine oxide, were detected in relatively small quantities. These peptides results likely from the oxidation of their not reported precursors: CLD' (c (Pro- Phe-Phe-Trp-Ile-Met-Leu-Leu)), CLE'(c (Pro-Leu-Phe-Ile-Met-Leu-Val-Phe)), CLF (c (Pro-Phe-Phe-Trp- Val-Met-Leu-Met), and CLG (c (Pro-Phe-Phe-Trp-Ile-Met-Leu-Met), present at higher concentrations in the extract protected from atmospheric oxygen. The sequences of the unreported cyclic peptides were proposed on the basis of CID experiments and homology with peptides described by Morita,1,2 and supported by the fragmentation of synthetic analogues of CLA of a known structure.     

Structural characterization of sulfonated azo dyes using liquid secondary ion mass spectrometry/tandem mass spectrometry

Eight monosulfonated and disulfonated azo dyes were analyzed using liquid secondary ion mass spectrometry/tandem mass spectrometry, in the negative ion mode, under low-energy collision conditions (110–150 eV). Many structurally characteristic fragment ions were obtained, several of which have not been reported previously using other mass spectrometric techniques. Among the structurally important ions observed were those due to loss of SO₂, SO₃ and various aromatic substituents, such as NO₂ and NHCOCH₃. Losses of N₂ were also proposed. In addition, product ions due to cleavage at the azo linkage were observed, and also SO₃^? and HSO₃^? ions. Several of the azo cleavage product ions detected did not contain sulfonate groups. Possible pathways for the formation of product ions are proposed. The structural information obtained should help to better characterize and identify sulfonated azo dyes in the future.     

Structural characterization of isoprenylated flavonoids from Kushen by electrospray ionization multistage tandem mass spectrometry

Eighteen isoprenylated flavonoids (8 flavanones, 3 flavanols, and 7 chalcones) isolated from Kushen or synthesized were studied by positive and negative ion electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Plausible fragmentation patterns were obtained by comparing their MS(n) spectra with each other, which were further supported by high-resolution MS data and two model compounds. It was shown that the 2'-OH group would make the C-ring of flavonoids studied more labile through a six-membered mechanism, resulting in base peaks of (1,3)A+ (positive mode) and (1,4)A(-) (negative mode). In addition, the 2'-OH is also responsible for the neutral loss of water in (+)ESI/MS(2) of flavanones. The neutral loss of water (or methanol) in (-)ESI/MS(2) of flavanols was elucidated by a E2 elimination mechanism. Different relative abundances (RA) of (1,3)A(+) and S(+) in (+)ESI/MS(2) spectra were used to discriminate flavanones with their open-ring products, chalcones, since the equilibrium for flavanone<-->chalcone isomerization in ESI ion source could not be obtained in positive mode.     

Molecular species analysis of arachidonate containing glycerophosphocholines by tandem mass spectrometry

Carboxylate anions arising from collision-induced dissociation (CID) of the [M - 15]^- ion produced by fast atom bombardment (FAB) of glycerophosphocholine (GPCho) were previously shown to be produced in an abundance ratio of 1:3 for the carboxylic acids esterified at sn - 1 and sn - 2, respectively. This observation has been confirmed in a series of 13 synthetic GPCho molecular species. A good correlation was found between the isomeric purity of GPCho molecular species as determined by negative-ion FAB/CID analysis and the isomeric purity of the sn - 2 fatty acid using a phospholipase A₂ assay. Negative-ion FAB mass spectra of several 1-0-alkyl-2-acyl-GPCho molecular species were found to be similar to those of diacyl GPCho. However, the cm spectra from the major high-mass ions are different from those of the diacyl species in that the [M - 15]^- ion yields only one carboxylate anion and the [M - 86]^- undergoes a neutral loss of the sn - 2 carboxylic acid as a major decomposition product. These results suggest several rules useful for structural characterization of GPCho molecular species by negative-ion tandem mass spectrometry (MS/MS): (1) For diacyl species, the mass of the two carboxyl anions plus the mass of the GPeho backbone (minus a methyl group) must correspond to the mass of the [M - 15] anion; (2) for diacyl species there is a carboxylate anion ratio approximately 1:3 for the substituents at sn - 1 and sn - 2; and (3) for alkylether species, only one fatty acyl group is present, and the difference between the [M - 15] ion and the GPCho backbone (minus methyl) plus the fatty acyl group at sn - 2 corresponds to an alkylether substituent. (4) Assignment of ether-linked molecular species can be made from the [M - 86]^- ion, which has a strong neutral loss of the sn - 2 fatty acid. Analysis of GPCho isolated from human neutrophils by total lipid extraction and normal-phase HPLC was carried out by negative-ion FABand MS/MS. The major arachidonate-eontaining molecular species, which comprise only 5% of total GPCho, were identified by using precursor ion scans for the arachidonate anion, m/ z 303. Decomposition of identified. precursor ions permitted the assignment of those molecular species of GPCho that contain arachidonate at sn - 2 and identification of the substituent at the sn - 1 position. These results were compared to previously identified molecular species from human neutrophils. Several minor arachidonate-containing molecular species were tentatively identified.     

Top-down characterization of proteins and drug-protein complexes using nanoelectrospray tandem mass spectrometry

We report a 'top-down' approach for characterization of proteins, and identification of binding sites in protein-drug complexes using nanoelectrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry (nanoESI-MS/MS). The efficiency of direct fragmentation of intact protein ions and the feasibility of this method were initially demonstrated using several well-characterized proteins with different molecular weights including metallothionein (6126 Da), cytochrome c (horse, 12360 Da), myoglobin (horse, 16592 Da), and hemoglobin (human, 64453 Da). Simply varying collision energy without enzyme digestion and gel or LC separation generated a range of peptide fragments of these proteins. Over 80% of these peptide ions matched those in the SWISS-PROT database with mass accuracy of 8 to 32 ppm with external calibration. This technique was further applied to fragment a cisplatin-metallothionein complex to identify the binding sites, demonstrating a potential application in the study of drug-protein binding.     

Electrospray tandem mass spectrometry for structural characterization of aporphine- benzylisoquinoline alkaloids

Electrospray mass spectrometry and tandem mass spectrometry techniques were utilized to elucidate the structures of ten aporphine-benzylisoquinoline alkaloids, consisting of monoether link between aporphine and benzyltetrahydroisoquinoline units, which were isolated and identified previously from a variety of Thalictrum sp. (Ranunculaceae family) based mainly on the UV, IR, CD, NMR, EI-MS, CI-MS, derivatization, and chemical degradation techniques. In this investigation, protonated molecules, [M+H]+ ions, for nine tertiary alkaloids, a molecular ion, [M+'] ion, for a quaternary alkaloid, and very intense doubly- protonated molecules, [M+2H]2+ ions (100% of relative abundance) in Q1 Scan MS spectra, and prominent as well as diagnostic product ions for structural information in the tandem MS/MS spectra were observed for all investigated alkaloids each in nanogram quantities. More than 10 microg quantities of each investigated alkaloid or other isoquinoline and aporphine analogs needed for the CI-MS, EI-MS and FAB-MS analysis from the previous studies.     

Electron impact ionization mass spectrometry and tandem mass spectrometry of phenylalkylpyridazines

The mass spectrometric fragmentation behaviour of pyridazine and four monosubstituted derivatives containing a pbenylalkyl side-chain (3- and 4-benizylpyridazine, 3- and 4-(2-pbenylethyl)pyridazine) was investigated. In the electron impact ionization mess spectra of the 3-substituted compounds abundant [M – H]⁺ peaks are observed. This allows a clear distinction between 3- and 4-substituted pyridazines, as the spectra of the latter isomers show only very weak [M – H]⁺ signals. The stability of [M – H]⁺ ions derived from 3-alkylpyridazines (deduced from only the very low abundance of further fragment ions) gives strong evidence for a cyclic structure of these ions. One fragmentation pathway typical of the parent pyridazine, the [M - N₂] fragmentation, was not detectable with any of the phenylalkylpyridazines investigated. Instead, loss of HCN, H₃CN⁺ and N²H⁺ was observed. An interesting fragmentation, observed with 3-(2-phenylethyl)pyridazine, is the loss of ⁺CH₃ from the molecular ion and also from the [M – H]⁺ ion.     

Electrospray mass spectrometry and tandem mass spectrometry of bimetallic oxovanadium complexes

A series of six bimetallic oxovanadium complexes (1-6; only one was purified) were investigated by electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) in negative-ion mode. Radical molecular anions [M](.-) were observed in MS mode. Fragmentation patterns of [M](.-) were proposed, and elemental compositions of most of the product ions were confirmed on the basis of the high-resolution ESI-CID-MS/MS spectra. A complicated series of low-abundance product ions similar to electron impact (EI) ionization spectra indicated the radical character of the precursor ions. Fragment ions at m/z 214, 200, and 182 seem to be the characteristic ions of bimetallic oxovanadium complexes. These ions implied the presence of a V-O-V bridge bond, which might contribute to stabilization of the radical. To obtain more information for structural elucidation, three representative bimetallic oxovanadium complexes (1-3) were analyzed further by MS in positive-ion mode. Positive-ion ESI-MS produced adduct ions of [M + H](+), [M + Na](+), and [M + K](+). The fragmentation patterns of [M + Na](+) were different than those of radical molecular anions [M](.-). Relatively simple fragmentation occurred for [M + Na](+), possibly due to even-electron ion character. Negative-ion MS and MS/MS spectra of the hydrolysis product of Complex 1 supported these finding, in particular, the existence of a V-O-V bridge bond.