Characteristic Isotope Patterns Research Articles

Hair protein and amino acids 13C and 15N abundances take more than 4 wk to reflect food isotopic patterns

Isotopic abundance (IA) of hair can be used as a biomarker for animal protein intake (PI) due to characteristic isotopic patterns of food sources (Petzke et al., J. Nutr. 135: 1515, 2005). In a controlled cross‐over study in 14 young women, we investigated if additional meat intake (200g pork fillet/d; M) or an omission of all meat and meat products (NOM) for 4 wk can influence natural stable nitrogen (15N) and carbon (13C) IA in bulk and amino acid (AA) specific hair, plasma and urine samples studied by IRMS. Daily PI (means ± SD) was 1.40 ± 0.29, 2.25 ± 0.35 or 1.15 ± 0.26 g/kg at baseline and in periods M and NOM, respectively. Hair and plasma proteins IA after 4 wk interventions did not change. Urinary IA were lower after period M compared to NOM (15N, P = 0.039; 13C, P = 0.006). The data suggest that a 4 wk period of altered meat intake is too short to be reflected in changed hair IA due to the high buffer capacity of endogenous protein mass and to preferential oxidation of excess dietary AA in the splanchnic bed. Interestingly, a 4 wk period of additional meat intake did not change fat free mass and plasma cholesterol‐, triglyceride‐, glucose‐, and homocysteine concentrations adversely compared to a meat‐free treatment.

Synthesis, characterisation and structures of thio-, seleno- and telluro-ether complexes of gallium(iii)

The reactions of GaX3 (X = Cl, Br or I) with SMe2, SeMe2 and TeMe2 (L) in non-coordinating solvents produces only the pseudo-tetrahedral [GaX3L], which have been characterised by IR, Raman and multinuclear NMR (1H, 71Ga, 77Se or 125Te) spectroscopy, and by the crystal structure of [GaCl3(SeMe2)]. The 71Ga NMR resonances show small low frequency shifts for fixed halides as the neutral donors change from S --> Se --> Te. Bidentate ligands including MeS(CH2)2SMe, PhS(CH2)2SPh, MeSe(CH2)2SeMe, nBuSe(CH2)2Se(n)Bu and MeTe(CH2)3TeMe (L-L) also produce complexes with 4-coordinate gallium centres, [(GaX3)2(mu-L-L)], confirmed by the crystal structures of [(GaI3)2(mu-MeS(CH2)2SMe)], [(GaCl3)2(mu-PhS(CH2)2SPh)] and [(GaCl3)2(mu-nBuSe(CH2)2Se(n)Bu)]. The structural data are consistent with the weaker Lewis acidity of the gallium as the halide co-ligands become heavier. Multinuclear NMR studies suggest that in chlorocarbon solutions partial dissociation of the ligands occur, which increases with the halide co-ligand Cl < Br < I. The o-xylyl dithioether, o-C6H4(CH2SMe)2, despite being pre-organised for chelation, also forms [(GaCl3)2(mu-L-L)]. The corresponding diselenoether complex decomposes in solution with C-Se bond cleavage to form the selenonium salt [o-C6H4CH2Se(Me)CH2][GaCl4], which was structurally characterised. The ditelluroether o-C6H4(CH2TeMe)2 undergoes rapid C-Te bond fission and rearrangement upon reaction with GaCl3, and the telluronium species [o-C6H4CH2Te(Me)CH2]+ and [MeTe(CH2(o-C6H4)CH2TeMe)2]+ have been identified by ES+ mass spectrometry from their characteristic isotope patterns.

Analysis of a chlorosulfolipid from Ochromonas danica by matrix‐assisted laser desorption/ionization quadrupole ion trap time‐of‐flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) is an established tool for analyzing high mass molecules, such as proteins, whereas it attracts far less interest in the field of lipid analysis. In the study reported here a new chlorosulfolipid (CSL), 3,8,12,15-tetrachloroeicosane-1,17,18-triyl tris(hydrogen sulfate), was identified from the alga Ochromonas danica and de novo characterized by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-ToF) MS in negative ion mode. This method provides an effective alternative for the analysis of compounds directly derived from organic cell extracts. For MALDI analyses several frequently used solid MALDI matrices as well as some ionic liquid matrices (ILMs) were tested to enhance the analyte response to UV-laser and its ionization. The molecular weight of the observed compound could be determined as Li-, Na- and K-adducts [M+Me-2H]-. The characteristic isotopic patterns of the measured ions and the well-allocated molecular fragments by MS1, MS2 and MS3 indicate the fourfold chlorination and threefold sulfation of the investigated compound. The MS fragmentation alongside of the chlorine-bearing C-atoms is accompanied by the generation of a double bond at the opposite fragment in MS1. This obtained fragmentation pattern provides an insight into the allocation of the chlorine-bearing C-atoms along the carbon chain.

ICP-MS-assisted nanoHPLC-electrospray Q/time-of-flight MS/MS selenopeptide mapping in Brazil nuts

A method was developed for the identification of selenium-containing peptides issued from proteins of a Se-rich (82.9 mg kg–1) foodstuff, Brazil nut (Bertholletia excelsa). A sample purification procedure was optimized to cope with the 100-fold excess of sulfur analogues and matrix interferences. It was based on the consecutive size-exclusion fractionation of proteins and tryptic peptides, and enrichment of the Se-containing fractions, prior to nanoHPLC-ES-Q/TOF MS/MS. The characteristic isotopic patterns of selenium compounds (always minor peaks) were detected in ESI mass spectra at retention times precisely indicated by the matrix interference-free, sensitive (DL 1.3 fmol) 80Se detection by ICP collision cell MS in the same separation conditions. The potential of the method was demonstrated by the identification of 15 Se-containing peptides, from which all but one were found to originate from the selenised isoforms of the 2S protein.

NIR Lanthanide Luminescence by Energy Transfer from Appended Terpyridine–Boradiazaindacene Dyes

Mononuclear trivalent lanthanide complexes with formula [Ln(L)(NO(3))(3)] [in which L=4,4-difluoro-8-(2':2'';6'':2'''-terpyridin-4''-yl)-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene (Boditerpy)] are reported for Ln=Yb, Nd, Er, La and Gd. According to the crystal structure of the Yb complex, the lanthanide ion is bound to the terdentate terpyridine and the inner coordination sphere of the nine-coordinate lanthanide ion is completed by three bidentate nitrate anions. The coordination polyhedron can be described as a distorted tricapped antiprism. The terpyridine chelate is almost planar and tilted by nearly 60 degrees from the indacene subunit. FT-IR spectra confirm the bidentate binding mode of the nitrate anions for the other complexes. NMR and ES-MS spectra (through characteristic isotopic patterns) confirm the chemical formulation. The complexes have high molar absorption coefficients in the visible spectral region (65,000 M(-1) cm(-1) at 529 nm) and display sizeable NIR luminescence (900 to 1600 nm, for Ln=Yb, Nd and Er), upon irradiation through the electronic state of the indacene moiety at 514 nm. Crystal-field splitting was analysed at low temperature. The quantum yield of the Yb solution (10(-4) M) in dichloromethane amounts to 0.31 %, corresponding to a sensitisation efficacy of the ligand of ca. 63 %.

Cyclic Polyvanadates Incorporating Template Transition Metal Cationic Species: Synthesis and Structures of Hexavanadate [PdV6O18]4-, Octavanadate [Cu2V8O24]4-, and Decavanadate [Ni4V10O30(OH)2(H2O)6]4-

Three types of heteropolyvanadates, [(C2H5)4N]4[PdV6O18] (1), [(C2H5)4N]4[Cu2V8O24] (2), and [(C6H5)4P]4[Ni4V10O30(OH)2(H2O)6] (3), were synthesized through the reaction between the [VO3]- anion and metal template cations of Pd(II), Cu(II), and Ni(II). The X-ray crystal structures of 1 (a = 29.952(4) A, b = 12.911(2) A, and c = 13.678(2) A, orthorhombic, space group Pca2(1) with Z = 4), 2 (a = 13.740(1) A, b = 22.488(2) A, c = 18.505(2) A, and beta= 94.058(2) degrees , monoclinic, space group P2(1)/n with Z = 4), and 3 (a = 12.333(2) A, b = 16.208(4) A, c = 16.516(3) A, alpha = 112.438(3) degrees , beta = 94.735(3) degrees , and gamma = 104.749(3) degrees , triclinic, space group P with Z = 1) demonstrate that the metal cationic species induced cyclic [VO3](n-)n (n = 6, 8, 10) ring formation and the cations are incorporated in the rings themselves. In the metal inclusion products, the cyclic vanadates act as macrocyclic ligands, in which the metal cationic species act as the templates. The cyclic vanadate is composed of tetrahedral VO4 units that share corners and incorporates a metal cationic species in the center of the molecules. The bowl-shaped complex 1 includes a Pd2+ cation that is coordinated by the oxygen donors of a boatlike hexavanadate ring. The diamagnetic complex 1 was characterized via 51V and 17O NMR spectroscopy. Complex 2 involves an octavanadate ring and two Cu2+, which are located on both sides of the mean plane as defined by the eight oxygen atoms that bridge the vanadium atoms. In the case of complex 3, the di-mu-hydroxo-bridged Ni2+ dimer with capped Ni2+ aqua ions is formed by hydrolysis to form the decavanadate ring, in which two of the tetrahedral vanadate units are not bonded to the Ni2+ core but supported by hydrogen bonds through the aqua-ligand in the capped Ni2+ cation. Complexes 1-3 in solution were clearly identified by their characteristic isotope patterns using ESI-MS studies.

Enhancing TOF/TOF-based de Novo Sequencing Capability for High Throughput Protein Identification with Amino Acid-Coded Mass Tagging

Because of the intrinsic physical properties of single- or double-charged ions, MALDI-based CID on these peptide precursor ions tends to be incomplete, resulting in a large number of MS/MS spectra unassigned or ambiguously identified. Consequently, the TOF/TOF high throughput capability may not be fully explored and utilized. Here, we describe a novel method for de novo sequence assignment of those MALDI TOF/TOF MS/MS spectra with incomplete or weak fragment ion series. In this approach, the deuterium-labeled lysine and leucine precursors were used in parallel to mass-tag the proteome of a metastatic human hepatocellular carcinoma (HCC) cell line during in vivo cell culturing. These stable isotope precursor markers not only position at terminal but at internal MS/MS fragment ions with the characteristic isotope pattern induced by multiple mass tagging in parallel. This enhanced signal specificity evidently resolved ambiguities in those sparse poor-quality TOF/TOF spectra by providing critical sequential links among MS/MS fragment ions. Our data-dependent approach was able to reduce many false-positives in current genome sequence-based peptide sequencing. With developing new algorithms accordingly, our approach is amenable for automation that will lead to more comprehensive and reliable identification for proteomes.

Highly luminescent, triple- and quadruple-stranded, dinuclear Eu, Nd, and Sm(III) lanthanide complexes based on bis-diketonate ligands.

The bis(beta-diketone) ligands 1,3-bis(3-phenyl-3-oxopropanoyl)benzene, H(2)L(1) and 1,3-bis(3-phenyl-3-oxopropanoyl) 5-ethoxy-benzene, H(2)L(2), have been prepared for the examination of dinuclear lanthanide complex formation and investigation of their properties as sensitizers for lanthanide luminescence. The ligands bear two conjugated diketonate binding sites linked by a 1,3-phenylene spacer. The ligands bind to lanthanide(III) or yttrium(III) ions to form neutral homodimetallic triple stranded complexes [M(2)L(1)(3)] where M = Eu, Nd, Sm, Y, Gd and [M(2)L(2)(3)], where M = Eu, Nd or anionic quadruple-stranded dinuclear lanthanide units, [Eu(2)L(1)(4)](2-). The crystal structure of the free ligand H(2)L(1) has been determined and shows a twisted arrangement of the two binding sites around the 1,3-phenylene spacer. The dinuclear complexes have been isolated and fully characterized. Detailed NMR investigations of the complexes confirm the formation of a single complex species, with high symmetry; the complexes show clear proton patterns with chemical shifts of a wide range due to the lanthanide paramagnetism. Addition of Pirkle's reagent to solutions of the complexes leads to splitting of the peaks, confirming the chiral nature of the complexes. Electrospray and MALDI mass spectrometry have been used to identify complex formulation and characteristic isotope patterns for the different lanthanide complexes have been obtained. The complexes have high molar absorption coefficients (around 13 x 10(4) M(-1)cm(-1)) and display strong visible (red or pink) or NIR luminescence upon irradiation at the ligand band around 350 nm, depending on the choice of the lanthanide. Emission quantum yield experiments have been performed and the luminescence signals of the dinuclear complexes have been found to be up to 11 times more intense than the luminescence signals of the mononuclear analogues. The emission quantum yields and the luminescence lifetimes are determined to be 5% and 220 micros for [Eu(2)L(1)(3)], 0.16% and 13 micros for [Sm(2)L(1)(3)], and 0.6% and 1.5 micros for [Nd(2)L(1)(3)]. The energy level of the ligand triplet state was determined from the 77 K spectrum of [Gd(2)L(1)(3)]. The bis-diketonate ligand is shown to be an efficient sensitizer, particularly for Sm and Nd. Photophysical studies of the europium complexes at room temperature and 77 K show the presence of a thermally activated deactivation pathway, which we attribute to ligand-to-metal charge transfer (LMCT). Quenching of the luminescence from this level seems to be operational for the Eu(III) complex but not for complexes of Sm(III) and Nd(III), which exhibit long lifetimes. The quadruple-stranded europium complex has been isolated and characterized as the piperidinium salt of [Eu(2)L(1)(4)](2-). Compared with the triple-stranded Eu(III) complex in the solid state, the quadruple-stranded complex displays a more intense emission signal with a distinct emission pattern indicating the higher symmetry of the quadruple-stranded complex.

Derivatisation of peptides with osmium tetroxide, 2,2′-bipyridine: capillary electrophoretic and MALDI–TOF mass spectrometric study

Abstract Site-specific chemical modification is a useful technology in characterisation of proteins, but the number of chemical probes of the protein structure reacting with proteins under mild conditions in aqueous solutions is rather limited. Here we studied the reaction of osmium tetroxide, 2,2′-bipyridine (Os,bipy) with several peptides using capillary zone electrophoresis (CZE) and matrix-assisted laser desorption-ionisation–time-of-flight mass spectrometry (MALDI–TOF MS). Both techniques showed formation of a stable complex of Os,bipy with tryptophan residues. In CZE peaks with different migration times and UV-Vis spectra were observed. MALDI–TOF MS showed the formation of a product with characteristic isotopic pattern corresponding to the presence of osmium atom. Oxidation of cysteine and methionine side chains to cysteic acid and methionine sulfone by Os,bipy was detected by CZE and confirmed by MALDI–TOF and post-source decay (PSD) mass spectra. PSD showed specific shifts of molecular weights of the peptides and their fragments after the derivatisation. We believe that Os,bipy may become a useful agent in the characterisation of proteins.

Derivatisation of peptides with osmium tetroxide, 2,2$prime;-bipyridine: capillary electrophoretic and MALDI?TOF mass spectrometric study

Site-specific chemical modification is a useful technology in characterisation of proteins, but the number of chemical probes of the protein structure reacting with proteins under mild conditions in aqueous solutions is rather limited. Here we studied the reaction of osmium tetroxide, 2,2′-bipyridine (Os,bipy) with several peptides using capillary zone electrophoresis (CZE) and matrix-assisted laser desorption-ionisation–time-of-flight mass spectrometry (MALDI–TOF MS). Both techniques showed formation of a stable complex of Os,bipy with tryptophan residues. In CZE peaks with different migration times and UV-Vis spectra were observed. MALDI–TOF MS showed the formation of a product with characteristic isotopic pattern corresponding to the presence of osmium atom. Oxidation of cysteine and methionine side chains to cysteic acid and methionine sulfone by Os,bipy was detected by CZE and confirmed by MALDI–TOF and post-source decay (PSD) mass spectra. PSD showed specific shifts of molecular weights of the peptides and their fragments after the derivatisation. We believe that Os,bipy may become a useful agent in the characterisation of proteins.

A Zn–porphyrin complex contributes to bright red color in Parma ham

The Italian traditional dry-cured ham (Parma ham) shows a stable bright red color that is achieved without the use of nitrite and/or nitrate. In this study we examined the pigment spectroscopically, fluoroscopically and by using HPLC and ESI-HR-MASS analysis. Porphyrin derivative other than acid hematin were contained in the HCl-containing acetone extract from Parma ham. A strong fluorescence peak at 588 nm and a weak fluorescence peak at 641 nm were observed. By HPLC analysis the acetone extract of Parma ham was observed at the single peak, which eluted at the same time as Zn-protoporphyrin IX and emitted fluorescence. The results of ESI-HR-MS analysis showed both agreement with the molecular weight of Zn-protoporphyrin IX and the characteristic isotope pattern caused by Zn isotopes. These results suggest that the bright red color in Parma ham is caused by Zn-protoporphyrin IX.

Isotopically labeled crosslinking reagents: resolution of mass degeneracy in the identification of crosslinked peptides.

Mass spectrometry in three dimensions (MS3D) is a newly developed method for the determination of protein structures involving intramolecular chemical crosslinking of proteins, proteolytic digestion of the resulting adducts, identification of crosslinks by mass spectrometry (MS), peak assignment using theoretical mass lists, and computational reduction of crosslinks to a structure by distance geometry methods. To facilitate the unambiguous identification of crosslinked peptides from proteolytic digestion mixtures of crosslinked proteins by MS, we introduced double 18O isotopic labels into the crosslinking reagent to provide the crosslinked peptides with a characteristic isotope pattern. The presence of doublets separated by 4 Da in the mass spectra of these materials allowed ready discrimination between crosslinked and modified peptides, and uncrosslinked peptides using automated intelligent data acquisition (IDA) of MS/MS data. This should allow ready automation of the method for application to whole expressible proteomes.

Reduction of [(C5Me5)2Mo2O5] and [(C5Me5)2Mo2O4] in Methanol/Water/Trifluoroacetate Solutions Investigated by Combined On‐Line Electrochemistry/Electrospray‐Ionization Mass Spectrometry

AbstractComplexes [Cp*2Mo2O5] (Cp* = η5‐C5Me5) and [Cp*2Mo2O4] were investigated by combined on‐line electrochemical (EC) reduction and electrospray‐ionization mass spectrometry (ESI‐MS) techniques in a trifluoroacetic acid buffered water/methanol solution. The reduction products at the larger negative potentials are identical for both compounds. The studies reveal the existence of a wide range of previously unknown di‐ and trinuclear MoV, MoIV, MoIII, and mixed‐valence complexes that were identified on the basis of their masses and characteristic isotope patterns. The structures of the initial compounds and the product of electroreduction with m/z = 713−729 were supported by in situ MSn experiments that allowed the elucidation of the fragmentation pathway for the collision‐induced dissociation. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2003)

Anionic adducts of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The formation and decomposition (postsource decay, PSD) of anionic adducts in negative ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry have been studied. Chloride, a small inorganic anion, has been found to form stable anionic adducts with a variety of neutral oligosaccharides that can survive the MALDI process to give readily identifiable signals (with characteristic isotope patterns) allowing subpicomole detection in the best cases. The matrixes that can aid the formation of chloride adducts of oligosaccharides have gas-phase acidities lower than or close to that of HCl (1373 kJ/mol). In PSD experiments, precursor chloride adducts of oligosaccharides yield fragment ions that retain the charge on the sugar molecule rather than solely forming Cl-, and these fragments can provide structurally informative product ions. In negative ion MALDI, highly acidic oligosaccharides do not form adducts with chloride anions, but mildly acidic saccharides (e.g., containing a carboxylic acid group) form both deprotonated molecules and chloride adducts, and each may provide structural information concerning the oligosaccharide upon decomposition.

Single Peptide-Based Protein Identification in Human Proteome through MALDI-TOF MS Coupled with Amino Acids Coded Mass Tagging

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.

Studies on the Reduction of [(C5Me5)2Mo2O5] in Methanol/Water/Acetate Solutions by On‐Line Electrochemical Flowcell and Electrospray Mass Spectrometry

AbstractThe complex [Cp*2Mo2O5] (Cp* = η5‐C5Me5) and its electrochemical reduction products in acetic acid/acetate‐buffered (pH = 4.0) water/methanol solutions were investigated by combined electrochemical (EC) flowcell and on‐line electrospray ionization mass spectrometry (ESI‐MS). Mono‐, di‐, tri‐, and tetranuclear organometallic molybdenum oxides were identified in the starting solution. The effect of the relevant ESI‐MS parameters (ionic mode, heated capillary voltage, and heated capillary temperature) and of the concentration on the observed distribution of ions in the mass spectrometer was studied in order to minimize side reactions in the ESI chamber. It was verified that reduction in the ESI‐MS is undetectable under open‐circuit conditions. The on‐line electrochemical study revealed the potential‐dependent formation of previously unknown mono‐, di‐, tri‐, and tetranuclear MoV, MoIV, and mixed‐valence complexes. The compounds were identified by their characteristic isotope patterns and their ion trap MSn fragmentations. The observed formation potentials reflect the higher stability of the multinuclear species relative to the mononuclear ones with the same oxidation state. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2003)

Identification of oxidized methionine residues in peptides containing two methionine residues by derivatization and matrix-assisted laser desorption/ionization mass spectrometry.

Oxidation of methionine residues in peptides and proteins occurs in vivo or may be an artifact resulting from purification steps. We present a three step method for the localization of methionine sulfoxides in peptides with two methionine residues. In the first step, the N-terminus as well as other reactive side chain functions are blocked by acetylation. The resulting protected peptides are cleaved by cyanogen bromide. The cleavage does not occur at methionine sulfoxide but only at reduced methionine residues forming new amino termini. The newly formed amino group is then derivatized with a bromine containing compound in the last step of the procedure. The resulting peptide can easily be identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using both the characteristic isotope pattern of the halogen and the metastable loss of methanesulfenic acid from oxidized residues. This procedure allows the unequivocal localization of oxidized methionines even in complex peptide mixtures.

Derivatization for liquid chromatography/electrospray mass spectrometry: synthesis of tris(trimethoxyphenyl)phosphonium compounds and their derivatives of amine and carboxylic acids.

A simple method for the derivatization of primary amines and carboxylic acids with tris(trimethoxyphenyl)phosphonium (TMPP) reagents to enhance their detection by electrospray mass spectrometry (ESI-MS) has been developed. The synthesis of novel TMPP reagents and their stable isotopically labelled analogues is described. Through the use of stable isotopically labelled TMPP "tags", incorporation of a doublet (1:1, (1)H/(2)H or (12)C/(13)C) into the target molecule can be achieved, enabling the use of isotopic target analysis to detect compounds of unknown molecular weight but with a characteristic isotope pattern and accurate mass difference.

Epitope Diversity of N-Glycans from Bovine Peripheral Myelin Glycoprotein P0 Revealed by Mass Spectrometry and Nano Probe Magic Angle Spinning 1H NMR Spectroscopy

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most abundant constituent of myelin. Using monoclonal antibodies, the homophilic binding of the P0 glycoprotein was shown to be mediated via the human natural keller cell (HNK)-1 epitope (3-O-SO(3)H-GlcUA(beta1-3)Gal(beta1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-glycan carrying the HNK-1 epitope, present on bovine peripheral myelin P0 (Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960). In this study, we report on the structural characterization of the detectable glycoforms, present on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid- or biantennary complex-type structures, the variety of epitopes is remarkable. In addition to the 3-O-sulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. This indicates the activity of a 6-O-sulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the disialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of such an epitope diversity triggers questions related to their function and whether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.

Rapid detection of multiple chlorinated bis(bibenzyls) in bryophyte crude extracts using laser desorption/ionization time-of-flight mass spectrometry.

Chlorinated bis(bibenzyls) of the bazzanine type were detected in crude bryophyte plant extracts of Bazzania trilobata from different locations using laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry without addition of an additional chemical matrix. The degree of chlorination could be identified by the characteristic isotope patterns. These results suggest that these chlorinated compounds are not artefacts of an incidental occurrence, or of the sample preparation, but are genuine natural products produced by the liverwort or by an endosymbiotic metabolism.