Inclusion of a Phytomedicinal Flavonoid in Biocompatible Surface-Modified Chylomicron Mimic Nanovesicles with Improved Oral Bioavailability and Virucidal Activity: Molecular Modeling and Pharmacodynamic Studies.

Morin hydrate (MH) is a widely-used Asian phytomedicinal flavonoid with a wide range of reported therapeutic activities. However, MH has limited oral bioavailability due to its low aqueous solubility and intestinal permeability, which in turn hinders its potential antiviral activity. The study reported herein was designed to encapsulate MH in polyethyleneglycolated (PEGylated) chylomicrons (PCMs) and to boost its antiviral activity and biological availability for oral administration using a rat experimental model. The PEGylated edge activator combined with the conventional components of chylomicrons (CMs) amplify the transport of the drug across the intestine and its circulation period, hence its therapeutic impact. The implementation of variables in the in vitro characterization of the vesicles was investigated. Using Design Expert® software, a 24 factorial design was conducted, and the resulting PCM formulations were fabricated utilizing a thin-film hydration technique. The efficacy of the formulations was assessed according to their zeta potential (ZP), entrapment efficiency percentage (EE%), amount of drug released after 8 h (Q8h), and particle size (PS) data. Formulation F9, which was deemed to be the optimal formula, used compritol as the lipidic core together in defined amounts with phosphatidylcholine (PC) and Brij52. Computer-aided studies revealed that MH alone in a suspension had both diminished intestinal permeability and absorption, but was enhanced when loaded in PCMs. This was affirmed by the superiority of formulation F9 results in ex vivo permeation and pharmacokinetic studies. Furthermore, formulation F9 had a superior safety profile and antiviral activity over a pure MH suspension. Molecular-docking studies revealed the capability of MH to inhibit MERS-CoV 3CLpro, the enzyme shown to exhibit a crucial role in viral replication. Additionally, F9 suppressed both MERS-CoV-induced histopathological alteration in lung tissue and resulting oxidative and inflammatory biomarkers. Collectively, the results reported herein affirmed the potential of PCMs as nanocarriers for the effective oral administration of MH as an antiviral.

Date palm wood as a new source of phenolic antioxidants and in preparation of smoked salmon.

The potential of date palm wood (DPW) as a new source of phenolic antioxidants was investigated in this contribution. The total phenolic content and antiradical activity of soluble and insoluble-bound fractions of DPW was compared to those of maple wood (MW). Furthermore, salmon was smoked with DPW and MW. Irrespective of the wood type, volatile phenolic compounds were mainly methoxyphenols, with the highest contribution from eugenol followed by guaiacol and their corresponding derivatives, as evaluated by solid-phase microextraction and gas chromatography-mass spectrometry. Salmon smoked with DPW showed a higher oxidative stability than that of MW during 21 days of storage at 4°C, which was explained by the higher content of volatile phenolic compounds in the smoke generated from DPW. Minor differences were detected for the instrumental color between both samples of smoked salmon. Therefore, smoking with DPW may be used for industrial meat and fish smoking purposes. PRACTICAL APPLICATIONS: Smoking has long been used to preserve fish and meat products. The process changes the appearance and gives a desirable flavor to the product. However, high temperatures applied during smoking may also induce lipid oxidation, the extent of which is counterbalanced by the antioxidant action of phenolics in woodsmoke. In this sense, the desired interactions of the smoke volatiles in the food matrix dictates the quality of the final product. Volatile phenolics released upon smoking are dependent on the type of wood. Thus, use of a specific wood might provide unique products, hence date palm wood (DPW) used in this work may not only provide special smoked fish products but its use could be extended to other smoked products. Hence, this contribution extends the possible feedstocks for the preparation of smoked products.

Structural investigation by tandem mass spectrometry analysis of a heterogeneous mixture of Lipid An isolated from the lipopolysaccharide of Aeromonas hydrophila SJ-55Ra.

RATIONALE: We report herein the electrospray ionization mass spectrometry (ESI-MS) negative ion mode and low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) analysis of a mixture of lipid An isolated from the lipopolysaccharide (LPS) of a rough-resistant wild strain of the Gram-negative bacteria Aeromonas hydrophila grown in the presence of phages (SJ-55Ra). This investigation indicates that the presence of a mixture of lipid A acylated disaccharides, whose molecular structures were not relatively conserved, resulted from the incomplete LPS biosynthesis caused by the phage treatment. METHODS: The heterogeneous lipid An mixture from the LPS-SJ55Ra was obtained following growth of the Gram-negative bacteria Aeromonas hydrophila (SJ-55R) in the presence of phages and isolation by the aqueous phenol method. Following hydrolysis and purification of the lipopolysaccharide, ESI-MS and low-energy CID-MS/MS analyses were performed on a triple-quadrupole (QqQ) and a Fourier transform ion cyclotron resonance (FTICR) instrument. RESULTS: ESI-MS analysis suggested that this lipid An mixture contained eight molecular disaccharide anions and three monosaccharide anions. This series of lipid An was asymmetrically substituted with ((R)-14:0(3-OH)) fatty acids located at O-3 and N-2 and with branched fatty acids: (Cl4:0(3-(R)-O-C14:0)) and (C12:0(3-(R)-O-(14:0)) at the O-3' and N-2' positions. CONCLUSIONS: Tandem mass spectrometric analyses allowed the exact determination of the fatty acid acylation locations on the D-GlcpN disaccharide. The MS/MS results established that it was possible to selectively cleave C-O, C-N, and C-C bonds, together with glycosidic C-O and cross-ring cleavages, affording excellent structural analysis of lipid A biomolecules.

The in situ gas-phase formation of a C-glycoside ion obtained during electrospray ionization tandem mass spectrometry. A unique intramolecular mechanism involving an ion-molecule reaction.

RATIONALE: This study examines the electrospray ionization mass spectrometry (ESI-MS), in-source collision-induced dissociation (CID) fragmentation and low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of a synthetic pair of ß- and α-anomers of the amphiphilic cholesteryl polyethoxy neoglycolipids containing the 2-azido-2-deoxy-D-galactosyl-D-GalN3 moiety. We describe the novel and unique in situ gas-phase formation of a C-glycoside ion formed during all these gas-phase processes and propose a reasonable mechanism for its formation. METHODS: The synthetic amphiphilic glycolipids were composed of the 2-deoxy-2-azido-D-galactosyl moiety (GalN3, the hydrophilic part) covalently attached to a polyethoxy spacer which is covalently linked to the cholesteryl moiety (hydrophobic part). The 2-azido-2-deoxy-α- and ß-D-galactosyl-containing glycolipids were studied by in-time and in-space ESI-MS and CID-MS/MS in positive ion mode, with quadrupole ion trap (QIT), quadrupole-quadrupole-time-of-flight (QqTOF), and Fourier transform ion cyclotron resonance (FTICR) instruments. RESULTS: Conventional single-stage ESI-MS analysis showed the formation of the protonated molecule. During the single-stage ESI-MS analysis and the CID-MS/MS of the [M+H](+) and [M+NH4](+) adducts obtained from both glycolipid anomers, the presence of a series of specific product ions with different intensities was observed, consistent with the [C-glycoside+H-N2](+), [cholestadiene+H](+), 2-deoxy-2-D-azido-galactosyl [GalN3](+), [GalNH](+) and [sugar-Spacer+H](+) ions. CONCLUSIONS: The gas-phase formation of the [C-glycoside+H-N2](+) ion isolated from the glycolipid anomers was observed during both the ESI-MS of the glycolipids and the CID-MS/MS analyses of the [M+H](+) ions and it was found to occur by an intramolecular rearrangement involving an ion-molecule complex. CID-QqTOF-MS/MS and CID-FTICR-MS(2) analysis allowed the differentiation of the two glycolipid anomers and showed noticeable variation in the intensities of the product ions.

Direct targeted glycation of the free sulfhydryl group of cysteine residue (Cys-34) of BSA. Mapping of the glycation sites of the anti-tumor Thomsen-Friedenreich neoglycoconjugate vaccine prepared by Michael addition reaction.

We present in this manuscript the characterization of the exact glycation sites of the Thomsen-Friedenreich antigen-BSA vaccine (TF antigen:BSA) prepared using a Michael addition reaction between the saccharide antigen as an electrophilic acceptor and the nucleophilic thiol and L-Lysine ε-amino groups of BSA using different ligation conditions. Matrix laser desorption ionization time-of-flight mass spectrometry of the neoglycoconjugates prepared with TF antigen:protein ratios of 2:1 and 8:1, allowed to observe, respectively, the protonated molecules for each neoglycoconjugates: [M + H](+) at m/z 67,599 and 70,905. The measurements of these molecular weights allowed us to confirm exactly the carbohydrate:protein ratios of these two synthetic vaccines. These were found to be closely formed by a TF antigen:BSA ratios of 2:1 and 8:1, respectively. Trypsin digestion and liquid chromatography coupled with electrospray ionization mass spectrometry allowed us to identify the series of released glycopeptide and peptide fragments. De novo sequencing affected by low-energy collision dissociation tandem mass spectrometry was then employed to unravel the precise glycation sites of these neoglycoconjugate vaccines. Finally, we identified, respectively, three diagnostic and characteristic glycated peptides for the synthetic glycoconjugate possessing a TF antigen:BSA ratio 2:1, whereas we have identified for the synthetic glycoconjugate having a TF:BSA ratio 8:1 a series of 14 glycated peptides. The net increase in the occupancy sites of these neoglycoconjugates was caused by the large number of glycoforms produced during the chemical ligation of the synthetic carbohydrate antigen onto the protein carrier.

Characterizing changes in snow crab (Chionoecetes opilio) cryptocyanin protein during molting using matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry.

RATIONALE: We report the matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) characterization of the cryptocyanin proteins of the juvenile Chionoecetes opilio crabs during their molting and non-molting phases. In order to assess the structural cryptocyanin protein differences between the molting and non-molting phases, the obtained peptides were sequenced by MALDI low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS). METHODS: The cryptocyanin protein was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by MALDI-TOF/TOF-MS. The purified cryptocyanin protein was sequenced, using the 'bottom-up' approach. After tryptic digestion, the peptide mixture was analyzed by MALDI-QqTOF-MS/MS and the data obtained were used for the peptide mass fingerprinting (PMF) identification by means of the Mascot database. RESULTS: It was demonstrated using MALDI-TOF/TOF-MS that the actual molecular weights of the non-molting and molting cryptocyanin proteins were different; these were, respectively, 67.6 kDa and 68.1 kDa. Using low-energy CID-MS/MS we have sequenced the trytic peptides to monitor the differences and similarities between the cryptocyanin molecular structures during the molting and non-molting stages. CONCLUSIONS: We have demonstrated for the first time that the actual molecular masses of the cryptocyanin protein during the molting and non-molting phases were different. The MALDI-CID-MS/MS analyses allowed the sequencing of the cryptocyanins after tryptic digestion, during the molting and non-molting stages, and showed some similarities and staggering differences between the identified cryptocyanin peptides.

High-energy collision-induced dissociation tandem mass spectrometry of regioisomeric lactose palmitic acid monoesters using matrix-assisted laser desorption/ionization.

RATIONALE: Structural characterization and differentiation of three newly synthesized lactose monopalmitate regioisomers at positions O-3, O-3' and O-6' were realized by single-stage matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) in the positive ion mode and by high-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS). METHODS: A MALDI-TOF/TOF analyzer was utilized for the analysis of these isobaric lactose monopalmitate regioisomers. The CID-MS/MS spectra were acquired using high-energy cid with a 2 kV potential difference between the source acceleration voltage and the collision cell. RESULTS: High-energy (CID) tandem mass spectrometry (MS/MS) analyses of the sodiated molecules, [M + Na](+), showed distinguishing cross-ring product ions and characteristic fingerprint product ions, which allowed the straight-forward mass spectrometric characterization of these different regiosiomers. CONCLUSIONS: This investigation allowed us to unravel the novel fragmentation behavior of the sodiated regioisoimer molecules obtained from the mono-substituted D-lactose fatty acid esters using high-energy CID-TOF/TOF-MS/MS analyses. The high-energy CID of the [M + Na](+) ions from the isobaric lactose monopalmitate regioiosmers promoted the formation of characteristic (0,2) A2 cross-ring cleavage product ions.

Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae, serotype Ogawa and the recombinant tetanus toxin C-fragment carrier.

We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C-fragment (rTT-Hc) carrier. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of neoglycoconjugates with carbohydrate : protein ratios of 1.9 : 1, 3.0 : 1, 4.0 : 1, 4.9 : 1, 5.9 : 1, 6.9 : 1, 7.9 : 1 and 9.1 : 1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate-protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3-D representation of the rTT-Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein.

Protein free microcapsules obtained from plant spores as a model for drug delivery: ibuprofen encapsulation, release and taste masking.

Sporopollenin exine capsules (SEC) extracted from Lycopodium clavatum spores were shown to encapsulate ibuprofen as a drug model, with 97 ± 1% efficiency as measured by recovery of the loaded drug and absence of the drug on the SEC surface by scanning electron microscopy (SEM). The encapsulated ibuprofen was shown to be unchanged from its bulk crystalline form by solid state NMR, FTIR and XRD. Essential for drug delivery applications, SEC were shown to be non-toxic to human endothelial cells and free of allergenic protein epitopes by MALDI-TOF-MS and ESI-QqToF-MS. Potential application for targeted release into the intestinal region of the gastrointestinal tract (GIT) was demonstrated by 88 ± 1% of the drug being retained in simulated gastric fluid (SGF) after 45 minutes and 85 ± 2% being released after 5 min in buffer (PBS; pH 7.4). The SEC were shown to provide significant taste masking of encapsulated ibuprofen in a double blind trial with 10 human volunteers.

Determination of glycation sites by tandem mass spectrometry in a synthetic lactose-bovine serum albumin conjugate, a vaccine model prepared by dialkyl squarate chemistry.

RATIONALE: Neoglycoconjugate vaccines synthesized by the squaric acid spacer method allow single point attachment of the carbohydrate antigen to the protein carrier. However, the localization of the carbohydrate antigen sites of conjugation on the protein carrier has been an elusive task difficult to achieve. METHOD: Covalent attachment of the lactose antigen to the bovine serum albumin (BSA) was prepared by the squaric acid method using a hapten:BSA ratio of 20:1. Different reaction times were used during the conjugation reaction and two different lactose-BSA glycoconjugate vaccines were obtained. The carbohydrate antigen hapten:BSA ratios of these lactose-BSA glycoconjugate vaccines were determined by MALDI-TOF/RTOF-MS and the glycation sites in the neoglycoconjugates were determined using nano-LC/ESI-QqTOF-MS/MS analysis of the trypsin and GluC V8 digests of the conjugates. RESULTS: We have identified a total of 15 glycation sites located on the BSA lysine residues for the neoglycoconjugate vaccine formed with a hapten:BSA ratio of 5.1:1, However, the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate with a hapten:BSA ratio of 19.0:1 allowed identification of 30 glycation sites located on the BSA. These last results seem to indicate that this conjugation results in formation of various glycoforms. CONCLUSIONS: It was observed that the number of identified glycation sites increased when the hapten:BSA ratio of glycoconjugate formation increased, and that the location of the glycation sites appears to be mainly on the outer surface of the BSA carrier molecule which is in line with the assumption that the sterically more accessible lysine residues, namely those located on the outer surface of the BSA, would be conjugated preferentially.

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry.

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.

Structural determination by atmospheric pressure photoionization tandem mass spectrometry of some compounds isolated from the SARA fractions obtained from bitumen.

We have identified compounds obtained from the SARA fractions of bitumen by using atmospheric pressure photoionization mass spectrometry and low-energy collision tandem mass spectrometric analyses with a QqToF-MS/MS hybrid instrument. The identified compounds were isolated from the maltene saturated oil and the aromatic fractions of the SARA components of a bitumen. The QqToF instrument had sufficient mass resolution to provide accurate molecular weight information and to enhance the tandem mass spectrometry results. The APPI-QqToF-MS analysis of the separated compounds showed a series of protonated molecules [M + H](+) and molecular ions [M](+▪) of the same mass but having different chemical structures, in the maltene saturated oil and the aromatic SARA fractions. These isobaric ions were a molecular ion [M2 ](+▪) at m/z 418.2787 and a protonated molecule [M5 + H](+) at m/z 287.1625 in the saturated oil fraction, and molecular ions [M6 ](+▪) at m/z 418.1584 and [M7 ](+▪) at m/z 287.1285 in the aromatic fraction. The identification of this series of chemical compounds was achieved by performing CID-MS/MS analyses of the molecular ions [M](+▪) ([M1 ](+▪) at m/z 446. 2980, [M2 ](+▪) at m/z 418.2787, [M3 ](+▪) at m/z 360.3350 and [M4 ](+▪) at m/z 346.2095) in the saturated oil fraction and of the [M5 + H](+) ion at m/z 287.1625 also in the saturated oil fraction. The observed CID-MS/MS fragmentation differences were explained by proposed different breakdown processes of the precursor ions. The presented tandem mass spectrometric study shows the capability of MS/MS experiments to differentiate between different classes of chemical compounds of the SARA components of bitumen and to explain the reasons for the observed mass spectrometric differences. However, greater mass resolution than that provided by the QqToF-MS/MS instrument would be required for the analysis of the asphaltene fraction of bitumen.

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O-PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry.

We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.

Determination of distinctive carbohydrate signatures obtained from the Aeromonas hydrophila (chemotype II) core oligosaccharide pinpointing the presence of the 4-O-phosphorylated 5-O-linked Kdo reducing end group using electrospray ionization quadrupole orthogonal time-of-flight mass spectrometry and tandem mass spectrometry.

The electrospray quadrupole orthogonal time-of-flight mass spectrometric (ESI-QqTOF-MS) structural elucidation of the core oligosaccharide of Aeromonas hydrophila (chemotype II) lipopolysaccharide has been investigated and it was demonstrated that it contained an 4-O-phosphorylated Kdo reducing end group, which was glycosylated by the remaining outer core oligosaccharide through its O-5 position. After releasing the core oligosaccharide from the native LPS with acid, the phosphorylated Kdo residue eliminated phosphoric acid, to produce a core oligosaccharide containing a mixture of diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids and an open-chain olefinic Kdo residue. The characteristic glycone sequence was elucidated by collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of the protonated molecule of the native core oligosaccharide. In addition, the analysis of the Hakomori permethylated core oligosaccharide was carried out by electrospray ionization quadrupole orthogonal time-of-flight mass spectrometry (ESI-QqTOF-MS) and matrix-assisted laser desorption/ionization (MALDI)-QqTOF-MS analyses. The presence of more than nine isobaric isomers of this core was detected. The CID-MS/MS analysis of the various protonated permethylated core oligosaccharide molecules showed a similar and diagnostic fragmentation pattern. The over-methylation of the permethylated core oligosaccharide containing either the 4,7- or the 4,8-anhydro-alpha-keto acid unit and the open-chain olefinic Kdo unit was reported. It was realized that the extra minor satellite signals obtained in the ESI-QqTOF-MS and MALDI-TOF-MS analyses were dimethyl sulfoxide (DMSO) stable covalent addition products, which have occurred by a Michael addition on the 4,8-Kdo exocyclic double bond. The occurrence of this series of covalent addition products during the MS analysis of a permethylated core oligosaccharide should be considered as 'carbohydrate-distinctive signatures' establishing and confirming the presence of a 4-O-phosphorylated-5-O-linked Kdo reducing end group.

Structural investigation of bacterial lipopolysaccharides by mass spectrometry and tandem mass spectrometry.

Mass spectrometric studies are now playing a leading role in the elucidation of lipopolysaccharide (LPS) structures through the characterization of antigenic polysaccharides, core oligosaccharides and lipid A components including LPS genetic modifications. The conventional MS and MS/MS analyses together with CID fragmentation provide additional structural information complementary to the previous analytical experiments, and thus contribute to an integrated strategy for the simultaneous characterization and correct sequencing of the carbohydrate moiety.

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides by both electrospray ionization and matrix-assisted laser desorption ionization QqToF tandem mass spectrometry.

The protein tropomyosin (TM) is a known major allergen present in shellfish causing frequent food allergies. TM is also an occupational allergen generated in the working environment of snow crab (Chionoecetes opilio) processing plants. The TM protein was purified from both claw and leg meats of snow crab and analyzed by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) using hybrid quadruple time-of-flight tandem mass spectrometry (QqToF-MS). The native polypeptide molecular weight of TM was determined to be 32,733 Da. The protein was further characterized using the 'bottom-up' MS approach. A peptide mass fingerprinting was obtained by two different enzymatic digestions and de novo sequencing of the most abundant peptides performed. Any post-translational modifications were identified by searching their calculated and predicted molecular weights in precursor ion spectra. The immunological reactivity of snow crab extract was evaluated using specific antibodies and allergenic reactivity assessed with serum of allergic patients. Subsequently, a signature peptide for TM was identified and evaluated in terms of identity and homology using the basic local alignment search tool (BLAST). The identification of a signature peptide for the allergen TM using MALDI-QqToF-MS will be critical for the sensitive and specific quantification of this highly allergenic protein in the work place.

Quantification of Greenland halibut serum vitellogenin: a trip from the deep sea to the mass spectrometer.

This paper focuses on the sequential steps involved in developing a technique for quantifying Greenland halibut vitellogenin, a serum protein biomarker, using a comprehensive mass spectrometric approach. In the first phase of this study, in-gel trypsin digestions of serum proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A characteristic band around a molecular mass of 185 kDa, present in the mature female specimens, but absent in the male samples, was identified as vitellognin according to the peptide mass fingerprint obtained by MALDI-MS. Subsequently, MALDI and electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses were performed on the digest of the vitellogenin band for de novo sequencing. From these studies, a characteristic 'signature' peptide (sequence: FFGQEIAFANIDK) was selected from a list of candidate peptides as a surrogate analytical standard used for quantification purposes. Sample preparation for vitellogenin quantification consisted of a simple one-step overnight trypsin digestion. Samples were spiked with an isotopologue signature peptide standard and analyzed by high-performance liquid chromatography (HPLC) coupled in-line to an electrospray quadrupole-hexapole-quadrupole tandem mass spectrometer, operated in selective reaction monitoring mode. Transitions [(m/z 750.0 --> 1020.4 and 750.0 --> 1205.4) and (754.8 --> 1028.6 and 754.8 --> 1213.2)] were monitored for the signature peptide and the internal standard, respectively. Samples obtained from the field showed that vitellogenin levels were in accordance with fish maturity determined by macroscopic examination of the gonad, proving this technique suitable for measuring vitellogenin as a serum protein biomarker for reproductive maturity in female fish.

Electrospray tandem mass spectrometric analysis of novel synthetic quinoxalinone derivatives.

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) using a hybrid QqToF-MS/MS instrument has aided the structural characterization and differentiation of a novel series of medicinal synthetic 1-N-glycoside-quinoxalinone derivatives. These derivatives 7 and 8 are formed by an amino bond between the cyclic N-1 of the quinoxaline moiety and the C-6 position of a fully protected methyl or allyl alpha-D-mannofuranoside 3 and 4, and subsequent deprotection of the mannopyranoside moiety. In general the novel synthetic quinoxaline derivatives afforded the protonated molecules in ESI. The breakdown routes of the protonated molecules were rationalized by conducting low-energy CID-MS/MS analyses. In addition, re-confirmation of the various established fragmentation routes was achieved by conducting a series of ESI-CID-QqTof-MS/MS product ion scans on various selected precursor ions, which were initiated by CID in the atmospheric pressure/vacuum interface using a higher declustering potential. ESI-QqToF-MS/MS analysis has proven to be a specific and very sensitive method for the structural identification in the gas phase of these novel glycoquinoxalinamine derivatives.

Elucidation of the complex molecular structure of wheat straw lignin polymer by atmospheric pressure photoionization quadrupole time-of-flight tandem mass spectrometry.

Wheat straw lignin was extracted using the novel CIMV procedure which selectively separates the cellulose, hemicelluloses and lignin. Solid-state (13)C NMR experiments using cross polarization/magic angle spinning (CP/MAS) were carried out on the extracted wheat straw lignin and some structural indices were revealed. Atmospheric pressure photoionization mass spectrometry (APPI-MS) has proven to be a powerful analytical tool capable of ionizing small to large lignin oligomers, which cannot be ionized efficiently by atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The APPI mass spectra of the extracted wheat straw lignin were recorded in the positive and negative ion modes. Positive ion mode APPI-MS indicated the exact presence of 39 specific oligomeric ions. Negative ion APPI-MS indicated the additional presence of at least 18 specific oligomeric ions. The structural characterization of this novel and complete series of 57 specific related oligomers was achieved by calculating the exact molecular masses measured by high-resolution quadrupole time-of-flight mass spectrometry (QqToF-MS). Some oligomeric species photoionized in both the positive and negative ion modes to form the respective protonated and deprotonated molecules. Low-energy collision-induced dissociation tandem mass spectrometric analyses performed with a QqToF-MS/MS hybrid instrument provided unique dissociation patterns of the complete series of novel precursor ions. These MS/MS analyses provided diagnostic product ions, which enabled us to determine the exact molecular structures and arrangement of the selected 57 different related ionic species.

Absolute quantification of Atlantic salmon and rainbow trout vitellogenin by the 'signature peptide' approach using electrospray ionization QqToF tandem mass spectrometry.

In this work, we present a simple method for absolute quantification of plasma vitellogenin from both rainbow trout and Atlantic salmon. Plasma samples obtained from control and beta-estradiol induced fish were digested with trypsin. A characteristic 'signature peptide' was selected and analyzed by high performance liquid chromatography (HPLC) coupled to an electrospray quadrupole-time-of-flight tandem mass spectrometer, using a deuterated homolog peptide as an internal standard. The hybrid tandem mass spectrometer was operated in a 'pseudo' selected reaction-monitoring mode in which three diagnostic product ions were monitored for identification and quantification purposes. The reproducibility (coefficient of variation approximately 5%) and sensitivity (limit of quantification (LOQ) of 0.009 mg/ml) achieved by this simple assay allow it to be considered as an alternative to immunological assays.