MS3 Spectra (ms3 + spectrum)

Distribution by Scientific Domains

Selected Abstracts

Oligosaccharide sequences in Quillaja saponins by electrospray ionization ion trap multiple-stage mass spectrometry

Susanna Broberg
Abstract Ten different samples with 13 previously identified saponin structures from Quillaja saponaria Molina were investigated by electrospray ionization ion trap multiple-stage mass spectrometry (ESI-ITMSn) in positive and negative ion modes. Both positive and negative ion mode MS1,MS4 spectra were analyzed, showing that structural information on the two oligosaccharide parts in the saponin can be obtained from positive ion mode spectra whereas negative ion mode spectra mainly gave information on one of the oligosaccharide parts. Analysis of MS1,MS4 spectra identified useful key fragment ions important for the structural elucidation of Quillaja saponins. A flowchart involving a stepwise procedure based on key fragments from MS1,MS3 spectra was constructed for the identification of structural elements in the saponin. Peak intensity ratios in MS3 spectra were found to be correlated with structural features of the investigated saponins and are therefore of value for the identification of terminal monosaccharide residues. Copyright 2004 John Wiley & Sons, Ltd. [source]

Protein glycosylation analysis by HILIC-LC-MS of Proteinase K-generated N - and O -glycopeptides

Gerhild Zauner
Abstract Analysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site-specific protein glycosylation is presented. Using asialofetuin and fetuin as model substances, a protocol for glycopeptide dissection was developed based on unspecific proteolysis by Proteinase K. The resulting glycopeptides were then resolved by nanoscale hydrophilic interaction liquid chromatography-electrospray multistage MS. The early elution range of O -glycopeptides was clearly separated from the late elution range of N -glycopeptides. Glycopeptides were analyzed by ion trap-MS/MS, which revealed fragmentations of glycosidic linkages and some peptide backbone cleavages; MS3 spectra predominantly exhibited cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N - and O -glycan attachment sites of fetuin could be confirmed; moreover using our method, the occupation of a new, additional O -glycosylation site serine 296 was found. In conclusion, this approach appears to be a valuable technique for in-depth analysis of the site-specific N -glycosylation and O -glycosylation of individual glycoproteins. [source]

Enantiomeric separation of mirtazapine and its metabolites by nano-liquid chromatography with UV-absorption and mass spectrometric detection

Salvatore Fanali
Abstract Mirtazapine (MIR) and two of its main metabolites, namely, 8-hydroxymirtazapine and N -desmethylmirtazapine, were separated in totheir enantiomers by nanoLC in a laboratory-made fused-silica capillary column (75 ,m ID) packed with a vancomycin-modified silica stationary phase. The simultaneous separation of the three couples of the studied enantiomers was achieved in less than 33 min, using an experimentally optimized mobile phase delivered in the isocratic mode. Optimization of the mobile-phase composition was achieved by testing the influence of the buffer pH and concentration, the water concentration, the organic modifier type and concentration, and on the retention and resolution of the analytes. The optimum mobile-phase composition contained 500 mM ammonium acetate pH 4.5/water/MeOH/MeCN, 1 : 14 : 40 : 45 v/v/v/v. Using a UV detector at 205 nm, the method was validated studying several experimental parameters such as LOD and LOQ, intraday and interday repeatability, and linearity. Good results were achieved: LOD and LOQ were in the range 5,15 and 10,40 ,g/mL, respectively (the highest value was obtained for the DEMIR enantiomers); correlation coefficients, 0.9993,0.9999; the intraday and interday precision was acceptable (RSD < 2%) using an internal standard. The method was tested for the separation of the studied enantiomers in an extracted (solid-phase) serum sample spiked with standard racemic mixture of MIR and its two metabolites. Finally, the nanoLC system was connected to a mass spectrometer through a nanoelectrospray interface and the MS, MS2, and MS3 spectra were acquired showing the potential of the system used for characterization and identification of the separated analytes. [source]

Semi-quantitative and structural metabolic phenotyping by direct infusion ion trap mass spectrometry and its application in genetical metabolomics

Albert Koulman
The identification of quantitative trait loci (QTL) for plant metabolites requires the quantitation of these metabolites across a large range of progeny. We developed a rapid metabolic profiling method using both untargeted and targeted direct infusion tandem mass spectrometry (DIMSMS) with a linear ion trap mass spectrometer yielding sufficient precision and accuracy for the quantification of a large number of metabolites in a high-throughput environment. The untargeted DIMSMS method uses top-down data-dependent fragmentation yielding MS2 and MS3 spectra. We have developed software tools to assess the structural homogeneity of the MS2 and MS3 spectra hence their utility for phenotyping and genetical metabolomics. In addition we used a targeted DIMS(MS) method for rapid quantitation of specific compounds. This method was compared with targeted LC/MS/MS methods for these compounds. The DIMSMS methods showed sufficient precision and accuracy for QTL discovery. We phenotyped 200 individual Loliumperenne genotypes from a mapping population harvested in two consecutive years. Computational and statistical analyses identified 246 nominal m/z bins with sufficient precision and homogeneity for QTL discovery. Comparison of the data for specific metabolites obtained by DIMSMS with the results from targeted LC/MS/MS analysis showed that quantitation by this metabolic profiling method is reasonably accurate. Of the top 100 MS1 bins, 22 ions gave one or more reproducible QTL across the 2 years. Copyright 2009 John Wiley & Sons, Ltd. [source]

Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition

Shengmin Sang
Tea is rich in polyphenols and has a variety of biological activities. In order to better understand the biological effects of tea constituents on human health, markers for their exposure and their metabolic fates are needed. Previously, we have characterized several catechin metabolites in the blood and urine, but more information on the metabolite profile of tea polyphenols is needed. In the present study, the human urinary metabolite profile of tea polyphenols was investigated using liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition. With data-dependent MS/MS analysis by collecting the MS2 and MS3 spectra of the most intense ions in the sample, we identified more than twenty metabolites of tea polyphenols from human urine samples. (,)-Epigallocatechin (EGC) glucuronide, methylated EGC glucuronide, methylated EGC sulfate, (,)-epicatechin (EC) glucruronide, EC sulfate, methylated EC sulfate, as well as the glucuronide and sulfate metabolites of the ring-fission metabolites of tea catechins, 5-(3,,4,,5,-trihydroxyphenyl)- , -valerolactone (M4), 5-(3,,4,-dihydroxyphenyl)- , -valerolactone (M6) and 5-(3,,5,-dihydroxyphenyl)- , -valerolactone (M6,), were the major human urinary metabolites of tea polyphenols. To our knowledge, this is the first report of the direct simultaneous analysis of the human urinary metabolite profile of tea polyphenols using single sample analysis. This method can also be used for thorough investigations of the metabolite profiles of many other dietary constituents. Copyright 2008 John Wiley & Sons, Ltd. [source]

Complementary structural information of positive- and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycans

Kisaburo Deguchi
Positive- and negative-ion MSn spectra of chicken egg yolk glycopeptides binding a neutral and a sialylated N-glycan were acquired by using electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) and collision-induced dissociation (CID) with helium as collision gas. Several characteristic differences were observed between the positive- and negative-ion CID MSn (n,=,2, 3) spectra. In the positive-ion MS2 spectra, the peptide moiety was presumably stable, but the neutral N-glycan moiety caused several B-type fragmentations and the sialylated N-glycan almost lost sialic acid(s). In contrast, in the negative-ion MS2 spectra, the peptide moiety caused several side-chain and N-glycan residue (e.g., N -acetylglucosamine (GlcNAc) residue) fragmentations in addition to backbone cleavages, but the N-glycan moieties were relatively stable. The positive-ion MS3 spectra derived from the protonated peptide ion containing a GlcNAc residue (203.1,Da) provided enough information to determine the peptide amino-acid sequence including the glycosylation site, while the negative-ion MS3 spectra derived from the deprotonated peptide containing a 0,2X1 -type cross-ring cleavage (83.1,Da) complicated the peptide sequence analysis due to side-chain and 0,2X1 residue related fragmentations. However, for the structural information of the N-glycan moiety of the glycopeptides, the negative-ion CID MS3 spectra derived from the deprotonated 2,4A6 -type cross-ring cleavage ion (neutral N-glycan) or the doubly deprotonated B6 -type fragment ion (sialylated N-glycan) are more informative than are those of the corresponding positive-ion CID MS3 spectra. Thus, the positive-ion mode of CID is useful for the analyses of peptide amino-acid sequences including the glycosylation site. The negative-ion mode of CID is especially useful for sialylated N-glycan structural analysis. Therefore, in the structural analysis of N-glycopeptides, their roles are complementary. Copyright 2006 John Wiley & Sons, Ltd. [source]