Multistage MS Research Articles

Monitoring of selected non-ionic surfactants in river water by liquid chromatography–tandem mass spectrometry

Alcohol ethoxylates (AEs) are a significant component of the non-ionic surfactant (NS) flux discharged into surface water. Due to the polydispersity of the majority of NS, they are easily recognizable by their ‘fingerprints’, i.e. a series of mass peaks which differ by m/z = 44, namely the m/z of a single oxyethylene subunit. Dodecanol ethoxylates (C12EOx) represent AEs from both renewable and petrochemical sources. Therefore, C12Ex are suitable fingerprints of NS in the aquatic environment.The aim of this work was to develop an LC–MS/MS method suitable for AE monitoring in river water.River water samples taken from the River Warta in Poznan (Poland) were extracted with ethyl acetate, evaporated, reconstituted in the mobile phase and processed by the LC – Multistage MS procedure (LC–MS/MS) using optimum multiple reaction monitoring (MRM). The method of multiple standard additions was used for the evaluation of each AE fingerprint concentration. The concentration of C12EOx having 2–9 oxyethylene subunits was determined. Standards for higher C12EOx are not yet available. The developed method offers an LOD of between 1 and 9 ng L−1, and is suitable for the monitoring of NS fingerprints in river water.The range of C12EO2–9 concentrations determined in the River Warta varied within two orders of magnitude in all cases. The lowest determined concentration was 17 ± 1 ng L−1, while the highest was 2.6 ± 0.14 μg L−1. The total concentration of C12EO2–C12EO9 homologues varied between 1.4 and 11.2 μg L−1.A relatively high concentration of short-chained homologues (2–5 oxyethylene subunits) was observed in the investigated river water. This provides evidence of a biodegradation pathway involving the gradual shortening of the AE oxyethylene chain. Distinct evidence was also obtained of unregulated NS discharges into the river.

Characterization of the designer drug deschloroketamine (2‐methylamino‐2‐phenylcyclohexanone) by gas chromatography/mass spectrometry, liquid chromatography/high‐resolution mass spectrometry, multistage mass spectrometry, and nuclear magnetic resonance

Clinical and forensic toxicology laboratories are challenged every day by the analytical aspects of the new psychoactive substances phenomenon. In this study we describe the analytical characterization of a new ketamine derivative, deschloroketamine (2-methylamino-2-phenylcyclohexanone), contained in seized powders. The analytical techniques employed include gas chromatography/mass spectrometry (GC/MS), liquid chromatography/electrospray ionization coupled with Orbitrap high-resolution/MS (LC/ESI-HRMS), multistage MS (ESI-MS(n)), and NMR. The LC/ESI-HRMS analyses consisted of accurate mass measurements of MH(+) ions in full-scan mode; comparison of experimental and calculated MH(+) isotopic patterns; and examination of the isotopic fine structure (IFS) of the M + 1, M + 2, M + 3 isotopic peaks relative to the monoisotopic M + 0 peak. The collision-induced product ions of the MH(+) ions were studied by both HRMS and MS(n). (1)H and (13)C NMR measurements were carried out to confirm the chemical structure of the analyte. The EI mass spectra obtained by GC/MS analysis showed the presence of molecular ions at m/z 203, and main fragment ions at m/z 175, 174, 160, 147, 146, and 132. The application of LC/ESI-HRMS allowed us to obtain: the accurate mass of deschloroketamine MH(+) ions with a mass accuracy of 1.47 ppm; fully superimposable experimental and calculated MH(+) isotopic patterns, with a relative isotopic abundance value of 3.69 %; and the IFS of the M + 1, M + 2, M + 3 isotopic peaks completely in accordance with theoretical values. Examination of the product ions of MH(+), as well as the study of both (1)H and (13)C NMR spectra, enabled the full characterization of the molecular structure of deschloroketamine. The combination of the employed analytical techniques allowed the characterization of the seized psychoactive substance, in spite of the lack of a reference standard. Deschloroketamine is a ketamine analogue considered to be more potent and longer lasting than ketamine, and this paper is probably the first to report on its analytical characterization.

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MSn)

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MSn, n = 2–4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M−H]− at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M−H]− ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides.

Primary Sequence Confirmation of a Protein Therapeutic Using Top Down MS/MS and MS(3).

Mass spectrometry has gained widespread acceptance for the characterization of protein therapeutics as a part of the regulatory approval process. Improvements in mass spectrometer sensitivity, resolution, and mass accuracy have enabled more detailed and confident analysis of larger biomolecules for confirming amino acid sequences, assessing sequence variants, and characterizing post translational modifications. This work demonstrates the suitability of a combined approach using intact MS and multistage top down MS/MS analyses for the characterization of a protein therapeutic drug. The protein therapeutic granulocyte-colony stimulating factor was analyzed using a Thermo Fusion Tribrid mass spectrometer using a multistage top down MS approach. Intact mass analysis identified the presence of two disulfide bonds based on exact mass shifts while a combined collision induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) MS/MS approach obtained 80% protein sequence coverage. Isolating MS/MS fragments for MS(3) analysis using HCD or CID increased the sequence coverage to 89%. 95% sequence coverage was obtained by reducing human granulocyte-colony stimulating factor (G-CSF) prior to MS/MS and MS(3) analysis to specifically target the residues between the disulfide bonds. The use of this combined intact MS and multistage top down MS approach allows for rapid and accurate determination of the primary sequence of a protein therapeutic drug product.

Chemical and Metabolic Profiling of Si-Ni Decoction Analogous Formulae by High performance Liquid Chromatography-Mass Spectrometry.

Along with an indispensable role in healthcare system of China for centuries, Traditional Chinese Medicine (TCM) shows increasing usages as complementary therapy in western countries. To improve our understanding on their therapeutic effects, it’s critical to unveil chemical compositions of TCM formula, the predominant form of therapy in TCM. However, intrinsic chemical complexity makes it a challenging task to perform analysis on each individual TCM formula even with most current state-of-art analytic techniques available. In this work we approached this question by focusing on analogous formulae, a unique category of TCM formulae grouped together based on shared herbs and/or similar TCM syndromes. Systematic chemical profiling on five Si-Ni decoctions (SNs) for cardiovascular diseases was performed by multistage MS and high-resolution MS (HR-MS) experiments. A total of 83 compounds, including alkaloids, flavonoids, ginsenosides, bile acids and triterpenoids, were described. Analysis on SNs-treated rats detected 55 prototype compounds and 39 metabolites in the systemic circulation in vivo, which may contribute directly to their observed clinical efficacies. This approach offers great advantage to speed up identification of chemical compositions of formula and reveal the difference among these analogous formulae that may be related to diverse clinical effects.

Identification of phenylpropanoids in Ciwujia injection by HPLC-MS

There are reports about the chemical compounds of Ciwujia herbs, but with no study report about the chemical material basis of Ciwujia injection (CWJI). In this study, LC-MS(n) and LC-Q-TOF-MS techniques were adopted for a qualitative analysis on phenylpropanoids in CWJI. The Ultmate XB-C18 column (4.6 mm x 250 mm, 5 microm) was adopted and eluted with the mobile phase of 0.5% formic acid-water and acetonitrile, with the flow rate at 0.8 mL x min(-1) and the column temperature at 20 degrees C. Based on the data of high-resolution and multi-stage MS, control products and literatures, altogether 54 phenylpropanoids were identified in Ciwujia Injection, including 34 phenylpropanoids, 16 ligans and 4 coumarins. Among them, 28 were reported for the first time in Ciwujia, and 14 compound structures were identified in comparison with the control products. The method established in this study could be used to simply and rapidly identify phenylpropanoids in CWJI. The findings provide scientific data for defining the chemical material basis of CWJI.

Multi‐stage tandem mass spectrometric analysis of novel β‐cyclodextrin‐substituted and novel bis‐pyridinium gemini surfactants designed as nanomedical drug delivery agents

This study aimed at evaluating the collision-induced dissociation tandem mass spectrometric (CID-MS/MS) fragmentation patterns of novel β-cyclodextrin-substituted- and bis-pyridinium gemini surfactants currently being explored as nanomaterial drug delivery agents. In the β-cyclodextrin-substituted gemini surfactants, a β-cyclodextrin ring is grafted onto an N,N-bis(dimethylalkyl)-α,ω-aminoalkane-diammonium moiety using variable succinyl linkers. In contrast, the bis-pyridinium gemini surfactants are based on a 1,1'-(1,1'-(ethane-1,2-diylbis(sulfanediyl))bis(alkane-2,1-diyl))dipyridinium template, defined by two symmetrical N-alkylpyridinium parts connected through a fixed ethane dithiol spacer. Detection of the precursor ion [M](2+) species of the synthesized compounds and the determination of mass accuracies were conducted using a QqTOF-MS instrument. A multi-stage tandem MS analysis of the detected [M](2+) species was conducted using the QqQ-LIT-MS instrument. Both instruments were equipped with an electrospray ionization (ESI) source. Abundant precursor ion [M](2+) species were detected for all compounds at sub-1 ppm mass accuracies. The β-cyclodextrin-substituted compounds, fragmented via two main pathways: Pathway 1: the loss of one head-tail region produces a [M-(N(Me)2-R)](2+) ion, from which sugar moieties (Glc) are sequentially cleaved; Pathway 2: both head-tail regions are lost to give [M-2(N(Me)2-R)](+), followed by consecutive loss of Glc units. Alternatively, the cleavage of the Glc units could also have occurred simultaneously. Nevertheless, the fragmentation evolved around the quaternary ammonium cations, with characteristic cleavage of Glc moieties. For the bis-pyridinium gemini compounds, they either lost neutral pyridine(s) to give doubly charged ions (Pathway A) or formed complementary pyridinium alongside other singly charged ions (Pathway B). Similar to β-cyclodextrin-substituted compounds, the fragmentation was centered on the pyridinium functional groups. The MS(n) analyses of these novel gemini surfactants, reported here for the first time, revealed diagnostic ions for each compound, with a universal fragmentation pattern for each compound series. The diagnostic ions will be employed within liquid chromatography (LC)/MS/MS methods for screening, identification, and quantification of these compounds within biological samples.

Specificity enhancement by electrospray ionization multistage mass spectrometry – a valuable tool for differentiation and identification of ‘V’‐type chemical warfare agents

The use of chemical warfare agents has become an issue of emerging concern. One of the challenges in analytical monitoring of the extremely toxic 'V'-type chemical weapons [O-alkyl S-(2-dialkylamino)ethyl alkylphosphonothiolates] is to distinguish and identify compounds of similar structure. MS analysis of these compounds reveals mostly fragment/product ions representing the amine-containing residue. Hence, isomers or derivatives with the same amine residue exhibit similar mass spectral patterns in both classical EI/MS and electrospray ionization-MS, leading to unavoidable ambiguity in the identification of the phosphonate moiety. A set of five 'V'-type agents, including O-ethyl S-(2-diisopropylamino)ethyl methylphosphonothiolate (VX), O-isobutyl S-(2-diethylamino)ethyl methylphosphonothiolate (RVX) and O-ethyl S-(2-diethylamino)ethyl methylphosphonothiolate (VM) were studied by liquid chromatography/electrospray ionization/MS, utilizing a QTRAP mass detector. MS/MS enhanced product ion scans and multistage MS(3) experiments were carried out. Based on the results, possible fragmentation pathways were proposed, and a method for the differentiation and identification of structural isomers and derivatives of 'V'-type chemical warfare agents was obtained. MS/MS enhanced product ion scans at various collision energies provided information-rich spectra, although many of the product ions obtained were at low abundance. Employing MS(3) experiments enhanced the selectivity for those low abundance product ions and provided spectra indicative of the different phosphonate groups. Study of the fragmentation pathways, revealing some less expected structures, was carried out and allowed the formulation of mechanistic rules and the determination of sets of ions typical of specific groups, for example, methylphosphonothiolates versus ethylphosphonothiolates. The new group-specific ions elucidated in this work are also useful for screening unknown 'V'-type agents and related compounds, utilizing precursor ion scan experiments.

Steroidal glycoalkaloid profiling and structures of glycoalkaloids in wild tomato fruit

Steroidal glycoalkaloids (SGAs) constitute one of the main groups of secondary metabolites in tomato fruit. However, the detailed composition of SGAs other than α-tomatine, dehydrotomatine and esculeoside A, remains unclear. Comparative SGA profiling was performed in eight tomato accessions, including wild tomato species by HPLC-Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FTICR/MS). On the basis of molecular formulae obtained from accurate m/z and fragmentation patterns by multistage MS/ MS (MS(n)), 123 glycoalkaloids in total were screened. Detailed MS(n) analysis showed that the observed structural diversity was derived from various chemical modifications, such as glycosylation, acetylation, hydroxylation and isomerization. Total SGA content in each tomato accession was in the range of 121-1986 nmol/gfr.wt. Furthermore, the compositional variety of SGA structures was distinctive in some tomato accessions. While most tomato accessions were basically categorized as α-tomatine-rich or esculeoside A-rich group, other specific SGAs also accumulated at high levels in wild tomato. Here, five such SGAs were isolated and their structures were determined by NMR spectroscopic analysis, indicating three of them were presumably synthesized during α-tomatine metabolism.

Fragmentation Behavior Studies of Chalcones Employing Direct Analysis in Real Time (DART)

Chalcones are naturally occurring, biologically active molecules generating interest from a wide range of researchapplications including synthetic methodology development, biological activity investigation and studying fragmentation patterns. Inthis article, a series of chalcones has been synthesized and their fragmentation behavior was studied using modern ambient ionizationtechnique Direct Analysis in Real Time (DART). DART ion source connected with an ion trap mass spectrometer wasused for the fragmentation of various substituted chalcones. The chalcones were introduced to the DART source using a glasscapillary without sample preparation step. All the chalcones showed prominent molecular ion peaks [M]•+ corresponding to thestructures. Multistage mass spectral data MSn (MS2 and MS3) were collected for all the chalcones studied. The chalcones withsubstitutions at 3, 4 or 5 positions gave product ion peaks with the loss of a phenyl radical (Ph•) by radical initiated α-cleavage,while substitution at 2 position of chalcone in the A-ring gave a product ion peak with the loss of substituted styryl radical(PhCH = CH•). In case of the chalcones with the substituent at 4 positions in A and B rings gave both types of fragmentation patterns. In conclusion, chalcones can be easily characterized using modern DART interface in very short time and efficientlywithout any cumbersome sample pretreatment.

Identification of an unusually sulfated tetrasaccharide chondroitin/dermatan motif in mouse brain by combining chip‐nanoelectrospray multistage MS2‐MS4 and high resolution MS

Chondroitin sulfate (CS)/dermatan sulfate (DS) are often found in nature as hybrid glycosaminoglycan chains in various proteoglycans. In the recent years, several MS methods were developed for the determination of over-, regular-, and undersulfated CS/DS chains. In the present work, the released hybrid CS/DS isolated and purified from mouse brain were digested with chondroitin AC lyase. The depolymerized chains were separated by gel filtration chromatography. Collected tetrasaccharides were analyzed by fully automated (NanoMate robot) chip-based nanoESI high capacity ion trap multistage MS (MS(2) -MS(4) ) recently introduced in glycosaminoglycan research by our laboratory. The obtained data were confirmed by high resolution MS screening and MS/MS performed on QTOF instrument. NanoMate-high capacity ion trap MS and QTOF MS screening revealed the presence in the mixture of oversulfated tetrasaccharides bearing three and four sulfate groups as well as traces of regularly and undersulfated hexamers. Additionally, several saturated species as either tetramers or hexamers exhibiting different sulfate content were discovered in the analyzed fraction. This diversity of the sulfation status indicates that the mouse brain might contain several types of proteoglycans. The molecular ions corresponding to trisulfated-[4,5Δ-GlcA-GalNAc-IdoA-GalNAc] were subjected to multistage fragmentation by CID. Sequence analysis data allowed for the postulation of two rare structural motifs: [4,5Δ-GlcA-GalNAc(4S)-IdoA(2S,3S)-GalNAc] and [4,5Δ-GlcA-GalNAc-IdoA(2S,3S)-GalNAc(4S)], previously not reported in neural tissue.

Identification tree based on fragmentation rules for structure elucidation of organophosphorus esters by electrospray mass spectrometry

Organophosphorus compounds have played important roles as pesticides, chemical warfare agents and extractors of radioactive material. Structural elucidation of phosphonates poses a particular challenge because their initial forms can be hydrolyzed, thus, degradation products may predominate in samples acquired in the field. The analysis of non-volatile organophosphorus compounds and their degradation products is possible using electrospray tandem mass spectrometry ESI-MS/MS. Here, we present a generic strategy that allows the unambiguous identification of substituents for two families of organophosphorus compounds: the phosphonates and phosphates. General fragmentation rules were deduced based on the study of decomposition pathways of 55 organophosphorus esters, including examples found in the literature. Multistage MS (MS(n)) experiments at high resolution in a hybrid mass spectrometer provide accurate mass measurements, whereas collision-induced dissociation experiments in a triple quadrupole give access to small fragment ions. The creation of a specific nomenclature for each possible structure of organophosphorus compound, depending on the alkyl side chain linked to the oxygen, was achieved by applying these fragmentation rules. This led to the creation of an 'identification tree' based upon the unique consecutive decomposition pathways uncovered for each individual compound. Hence, seven structural motifs were created that orient an unequivocal identification using the 'identification tree'. Despite the similar structures of the ensemble of phosphate and phosphonate esters, distinct identifications based upon characteristic neutral losses and diagnostic fragment ions were possible in all cases.

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti-inflammatory and hepatoprotective activities. Ultra-performance LC-quadrupole TOF-MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MS(n) methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC-MS(n). The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra-performance LC-MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long-term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.

Substructure‐based annotation of high‐resolution multistage MSn spectral trees

High-resolution multistage MS(n) data contains detailed information that can be used for structural elucidation of compounds observed in metabolomics studies. However, full exploitation of this complex data requires significant analysis efforts by human experts. In silico methods currently used to support data annotation by assigning substructures of candidate molecules are limited to a single level of MS fragmentation. We present an extended substructure-based approach which allows annotation of hierarchical spectral trees obtained from high-resolution multistage MS(n) experiments. The algorithm yields a hierarchical tree of substructures of a candidate molecule to explain the fragment peaks observed at consecutive levels of the multistage MS(n) spectral tree. A matching score is calculated that indicates how well the candidate structure can explain the observed hierarchical fragmentation pattern. The method is applied to MS(n) spectral trees of a set of compounds representing important chemical classes in metabolomics. Based on the calculated score, the correct molecules were successfully prioritized among extensive sets of candidates structures retrieved from the PubChem database. The results indicate that the inclusion of subsequent levels of fragmentation in the automatic annotation of MS(n) data improves the identification of the correct compounds. We show that, especially in the case of lower mass accuracy, this improvement is not only due to the inclusion of additional fragment ions in the analysis, but also to the specific hierarchical information present in the MS(n) spectral trees. This method may significantly reduce the time required by MS experts to analyze complex MS(n) data.

Isolation and characterization of flavonols from blackcurrant by high‐performance counter‐current chromatography and electrospray ionization tandem mass spectrometry

Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties of other minor flavonoids constituents have not yet been investigated due the difficulties related to their isolation. Multiple steps of high-performance counter-current chromatography in combination with ESI tandem mass spectrometry (MS(n)) were successfully used for the preparative isolation of flavonols from blackcurrant extract, to study their electrospray ionization mass spectrometry fragmentation behavior. Seven flavonols, namely myricetin-3-O-rutinoside (145.5 mg), myricetin-3-O-hexoside (79.7 mg), myricetin-3-O-(6″-malonyl)-glucoside (17.4 mg), kaempferol-3-O-glucoside (20.5 mg), quercetin-3-O-rutinoside (55.1 mg), quercetin-3-O-hexoside (25.8 mg), and myricetin (129.1 mg) have been successfully isolated and their multistage MS(n) data were used for detailed structure characterization. The results of these experiments demonstrated that high-performance counter-current chromatography along with ESI-MS(n) is a sensitive, selective, and effective technology for isolation and characterization of minor constituents from a complex mixture.

Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1)

Human LOX-1/OLR 1 plays a key role in atherogenesis and endothelial dysfunction. The N-glycosylation of LOX-1 has been shown to affect its biological functions in vivo and modulate the pathogenesis of atherosclerosis. However, the N-glycosylation pattern of LOX-1 has not been described yet. The present study was aimed at elucidating the N-glycosylation of recombinant human LOX-1 with regard to N-glycan profile and N-glycosylation sites. Here, an approach using nonspecific protease (Pronase E) digestion followed by MALDI-QIT-TOF MS and multistage MS (MS(3)) analysis is explored to obtain site-specific N-glycosylation information of recombinant human LOX-1, in combination with glycan structure confirmation through characterizing released glycans using tandem MS. The results reveal that N-glycans structures as well as their corresponding attached site of LOX-1 can be identified simultaneously by direct MS analysis of glycopeptides from non-specific protease digestion. With this approach, one potential glycosylation site of recombinant human LOX-1 on Asn(139) is readily identified and found to carry heterogeneous complex type N-glycans. In addition, manual annotation of multistage MS data utilizing diagnostic ions, which were found to be particularly useful in defining the structure of glycopeptides and glycans was addressed for proper spectra interpretation. The findings described herein will shed new light on further research of the structure-function relationships of LOX-1 N-glycan.

Identification of O-methyl-(−)-epicatechin-O-sulphate metabolites by mass-spectrometry after O-methylation with trimethylsilyldiazomethane

(−)-Epicatechin, an abundant dietary polyphenol found mainly in cocoa and tea, is known to extensively undergo metabolism after ingestion giving rise to a complex series of conjugated metabolites including numerous isomers. In the present study, the combination of fractionation, chemical derivatization and various mass spectrometric approaches is described to determine the exact position of sulphate group in methylated epicatechin metabolites. Four O-methyl-(−)-epicatechin-O-sulphate metabolites isolated from human urine samples were derivatized under mild condition using trimethylsilyldiazomethane (TMSD) in the presence of methanol. The resulting methylated reaction products were then analyzed by high resolution and multistage mass spectrometry for the subsequent identification of the sulphate positional isomers. Results show that O-methylation affects the charge delocalization in negatively charged ions and hereby the fragmentation pattern of the sulphate isomers allowing the identification of diagnostic ions. In addition, this study demonstrates that methoxy derivatives of polyphenol metabolites can be prepared using TMSD. Subsequently, the localization of the sulphate group in the polyphenol metabolites can be achieved by analyzing the methoxy derivatives by multistage mass spectrometry. Using an enzymatic reaction for identification of the O-methyl position, and a chemical O-methylation with TMSD follow by high resolution and multistage tandem MS for the identification of the sulphate group position, we were able to identify the previously unknown O-methyl-(−)-epicatechin-O-sulphate. Accordingly, we identified 3′-O-methyl-(−)-epicatechin-5-O-sulphate and 3′-O-methyl-(−)-epicatechin-7-O-sulphate as the main O-methyl-(−)-epicatechin-sulfates(−)-epicatechin metabolites in humans.

An invertebrate [hydroxyproline]-modified neuropeptide: Further evidence for a close evolutionary relationship between insect adipokinetic hormone and mammalian gonadotropin hormone family

An octapeptide of the adipokinetic hormone (AKH) peptide family is identified in the corpora cardiaca of the stink bug, Nezara viridula, by ESI-MSN (electrospray ionization multistage MS). This is the second AKH in N. viridula and it has a hydroxyproline residue at position 6, whereas the major AKH (known as Panbo-RPCH) has Pro as the sixth amino acid residue. The correct sequence assignment of [Hyp6]-Panbo-RPCH is confirmed by retention time and MS spectra of the synthetic peptide. Various extraction procedures were followed to ascertain whether the hydroxylation is an artefact of extraction, or whether it is due to a true post-translational modification at the prohormone level. The proline hydroxylation is unique for invertebrate neuropeptides, while it has been described in the vertebrate gonadotropin-releasing hormone (GnRH). The current finding is another piece of evidence that AKH and GnRH form a peptide superfamily and are closely related evolutionarily. Biologically, [Hyp6]-Panbo-RPCH is active in vivo as an AKH, causing hyperlipaemia in the stink bug at low doses, indicating again that it is an endogenous, mature and functional hormone in this insect species.

Sequencing cyclic peptides by multistage mass spectrometry

Some of the most effective antibiotics (e.g. Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of MS-based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage MS, and show its advantages over single-stage MS. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria.

Identification of benzo[ c]phenanthridine metabolites in human hepatocytes by liquid chromatography with electrospray ion-trap and quadrupole time-of-flight mass spectrometry

The metabolism of the benzo[ c]phenanthridine alkaloids was studied using human hepatocytes which are an excellent model system for biotransformation studies. For analysis of the alkaloids and their metabolites, an electrospray quadrupole ion-trap mass spectrometry (ESI ion-trap MS) connected to a reversed phase chromatographic system based on cyanopropyl modified silica was used. The optimized experimental protocol allowed simultaneous analysis of the alkaloids and their metabolites and enabled study of their uptake into and interconversion in human hepatocytes. The results show that formation of the dihydro metabolite which may be followed by specific O-demethylenation/ O-demethylation processes, is probably the main route of biotransformation (detoxification) of the benzo[ c]phenanthridines in human hepatocytes. The structure of the main O-demethyl metabolite (2-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridin-1-ol; 336.1 m/ z,) was proposed by the multi-stage MS and quadrupole time-of-flight MS methods using chemically synthesized standard.