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

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.     

Structural assignment of isomeric 2-aminopyridine-derivatized oligosaccharides using negative-ion MSn spectral matching

To investigate the possibility of structural assignment based on negative-ion MS2 spectral matching, three isomeric pairs of 2-aminopyridine (PA)-derivatized non-fucosylated, fucosylated, and sialylated oligosaccharides (complex type N-glycans) were analyzed using high-performance liquid chromatography/ion trap mass spectrometry (HPLC/ITMS) with a sonic-spray ionization (SSI) source. In the SSI negative-ion mode the deprotonated molecule [M-2H]2- becomes prominent. Negative-ion MS2 spectra derived from such ions contain many fragment types (B and Y, C and Z, A, and D) and therefore are more informative than the positive-ion MS2 spectra derived from [M+H+Na]2+ ions, which usually consist mainly of B and Y fragment ions. In particular the internal ions (D- and E-type ions) provided useful information about the alpha1-6 branching patterns and the bisecting GlcNAc residue. Spectral matching based on the correlation coefficients between negative-ion MS2 spectra was performed in a manner similar to the positive-ion MS2 spectral matching previously reported. It was demonstrated that negative-ion MS2 spectral matching is as useful and applicable to the structural assignment of relatively large non-fucosylated, fucosylated, and sialylated PA-oligosaccharide isomers as its positive-ion counterpart.     

Positive- and negative-ion chemical ionization gas chromatography/mass spectrometry of polynuclear aromatic hydrocarbons

Four groups of isomeric polynuclear aromatic hydrocarbons (PAH) were examined by gas chromatography/mass spectrometry (GC/MS) using positive-ion chemical ionization and negative-ion chemical ionization with a variety of reagent gases in order to evaluate the utility of each; differentiation of isomers was the ultimate goal. Hydrogen positive-ion chemical ionization (PICI) yielded different spectra for all but one isomer pair while retaining sensitivity comparable to electron-impact mass spectrometry. Several experimental conditions in the negative-ion mode afforded distinctly different spectra for isomeric PAH, but often sensitivities were reduced. The thirteen model compounds divided approximately into three classes according to the types and extent of reactions of the molecular anion. Class 1 gave as good sensitivity as hydrogen PICI; class 2 gave isomer-dependent spectra, but reduced sensitivity; class 3 gave no isomer differentiation, but greatly enhanced sensitivity.     

Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The use of post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the structural analysis of ((partly) methyl-esterified) oligogalacturonides (oligoGalA) is described. The fragmentation behavior of purified (un)saturated oligoGalA (degree of polymerization 3-6), methyl-esterified and methyl-glycosydated oligoGalA was studied. General fragmentation patterns are described and used for the elucidation of the positions of methyl esters on partly methyl-esterified oligoGalA. This technique now permits the determination of the position of methyl esters or other substituents on pectic fragments, helping in understanding the mode of action of pectinolytic enzymes.     

Differential fragmentation patterns of pectin oligogalacturonides observed by nanoelectrospray quadrupole ion-trap mass spectrometry using automated spectra interpretation

Oligogalacturonides of different degrees of polymerization (DP) and methyl esterification (DE) were structurally analyzed by nanoESI quadrupole ion-trap mass spectrometry. The fragmentation patterns of the oligogalacturonides were compared using the program 'Virtual Expert Mass Spectrometrist' (VEMS) for structural annotation. In the analyzed oligogalacturonides of lower DP, the generation of C/Y ions, i.e. ions retaining the glycosidic oxygen, was higher than that of B/Z ions. In general, with oligogalacturonides of higher DP, the B/Z ions were generated more abundantly. Oligogalacturonides with free carboxylic acid groups underwent higher water loss compared to fully methyl-esterified oligogalacturonides under the same fragmentation conditions. Cross-ring cleavage, in which fragmentation occurs across the ring system of the galacturonate residue and signified by unique mass losses, was observed to be higher in fully methyl-esterified oligogalacturonides than in non-methyl-esterified ones. This study demonstrates the different fragmentation patterns of oligogalacturonides as influenced by the presence or absence of methyl ester groups. For a detailed analysis of unknown oligogalacturonides, cross-ring fragmentation gives more structural information than glycosidic bond cleavage. One implication of this is that more structural information is obtained when analyzing methyl-esterified oligogalacturonides than non-methyl-esterified ones in an ion-trap instrument. This is of particular importance in pectin chemistry, where mass spectrometry has become the technique of choice for structural determination. Although this study was not designed to explain the mechanisms of oligogalacturonide fragmentation, possible explanations for why non-methyl-esterified oligogalacturonides undergo more water loss than methyl-esterified ones will be postulated. In addition, the VEMS program was extended to automatically interpret and assign the fragment ions peaks generated in this study.     

Positive ion formation in the ultraviolet matrix-assisted laser desorption / ionization analysis of oligonucleotides by using 2,5-dihydroxybenzoic acid

The development of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and its demonstrated performance with large proteins has generated substantial interest in utilizing this technique as an alternative to gel electrophoresis for DNA sequence analysis. However, a lack of understanding of the desorption and ionization processes has greatly hampered advances in this field. This article explores the formation of positively charged oligonucleotides in UV (355-nm) MALDI analysis by using the matrix 2,5-dihydroxybenzoic acid. Whereas substantial fragmentation is observed in the positive-ion mode by using the short oligomer d(TAGGT), no fragmentation is evident in the negative-ion mode under identical conditions. The fragmentation products are consistent with a previously published model in which base protonation initiates base loss, which leads to subsequent cleavage of the phosphodiester backbone. Several polydeoxythymidilic acids containing modified nucleosides were used to investigate positive-ion formation. The results support the hypothesis that positive ions are formed by protonation of the nucleobases. Because base protonation initiates base loss, fragmentation is intrinsic to positive-ion formation in the MALDI analysis of oligonucleotides. This result explains the dramatic difference in fragmentation observed in positive-ion compared to negative-ion UV-MALDI mass spectra of oligonucleotides.     

Negative-ion electrospray ionization tandem mass spectrometry of N-phosphoryl amino acids and dipeptides

The negative-ions of N-phosphoryl amino acids were studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The negative-ion ESI-MS/MS of N-phosphoryl amino acids showed characteristic fragmentation patterns different from those observed in the corresponding positive-ion ESI-MS/MS and negative-ion fast-atom bombardment mass spectra. For negative-ion ESI-MS/MS, a unique fragmentation from the N-terminal of N-phosphoryl amino acids or peptides containing a free beta-OH or CO(2)H group was observed to yield the characteristic fragment ion (RO)(2)P(O)O(-). The ease of the rearrangement depended on the position of the hydroxyl group in amino acids or peptides, and the N --> O rearrangement mechanism was proposed to involve the participation of the hydroxyl group. From previous solution-phase experiments and theoretical calculations, it was found that the beta-OH group was more active than gamma-OH, and the corresponding difference in negative-ion ESI-MS/MS was consistent with those previous findings.     

Characterization of phosphorylated amino acids by fast-atom bombardment mass spectrometry

Positive- and negative-ion fast-atom bombardment (FAB) mass spectrometry and linked-field scan techniques at constant B/E are used to characterize phosphorylated serine, threonine, and tyrosine amino acids. Abundant molecular ions are formed for all three amino acids in both modes of ionization. The dominant fragmentation is cleavage of the phosphate ester bond with charge retention in positive-ion FAB by the amino acid backbone and in the negative-ion mode by the phosphate group. The unique feature of positive-ion FAB mass spectra of phosphoserine and -threonine is the loss, from the ion [M + H]+, of a molecule of phosphoric acid (98 Da), whereas the corresponding tyrosine expels a HPO4 (96 Da) moiety to yield a stable phenylalanine ion.     

Direct analysis in real time (DART) mass spectrometry of nucleotides and nucleosides: elucidation of a novel fragment [C<Subscript>5</Subscript>H<Subscript>5</Subscript>O]<Superscript>+</Superscript> and its in-source adducts

Direct analysis in real time (DART) mass spectrometry is a recently developed innovative technology, which has shown broad applications for fast and convenient analysis of complex samples. Due to the ease of sample preparation, we have recently initiated an investigation of the feasibility of detecting nucleotides and nucleosides using the DART-AccuTOF instrument, which we will refer to as the DART mass spectrometer. Our experimental results reveal that the ions representing the intact molecules of nucleotides are not detectable in either positive-ion or negative-ion mode. Instead, all four natural nucleotides fragment in the DART ion source, and a common fragment ion, [C₅H₅O]⁺ (1), is observed, which is probably formed via multiple-elimination reactions. Interestingly, 1 can form adducts with nucleobases in different molar ratios in the DART ion source. In contrast to nucleotides, the ions representing the intact molecules of nucleosides are detected in both positive-ion and negative-ion mode using DART mass spectrometry. Surprisingly, the fragmentation pattern of nucleosides is different from that of nucleotides in the DART ion source. In the cases of nucleosides (under positive-ion conditions), the production of 1 is not observed, indicating that the phosphate group plays an important role for the multiple eliminations observed in the spectra of nucleotides. The in-source reactions described in the present work show the complexity of the conditions in the DART ion source, and we hope that our results illustrate a better understanding about DART mass spectrometry.     

Identification of amino acids by material enhanced laser desorption/ionisation mass spectrometry (MELDI-MS) in positive- and negative-ion mode

In the present study, different silica gel modifications were evaluated for their application as target surface for material enhanced laser desorption/ionisation mass spectrometric (MELDI-MS) investigation of amino acids. 4,4′-Azodianiline (ADA-silica) modified silica gel was successfully employed for the qualitative analysis of amino acids in positive- and in negative-ion mode. Further no derivatisation of amino acids was necessary, as the introduced system allowed the direct analysis of targets and delivered spectra with excellent signal intensity and signal-to-noise ratio within a few minutes. The influence of surface chemistry, ionisation mode and the nature of analytes on signal intensity was studied and discussed. Detection limit of 2.10 pg (10 fmol) was achieved by employing ADA-silica in positive-ion mode. Finally, xylem saps from different types of trees were analysed. This proved the high performance and excellent behaviour of the introduced target surface material.     

Structural analysis of 2,6-[bis(alkyloxy)methyl]-phenyltin derivatives using electrospray ionization mass spectrometry

Electrospray ionization (ESI) mass spectrometry was applied to the structural analysis of 23 2,6-[bis(alkyloxy)methyl]phenyltin(IV) derivatives. The mass spectra were measured in both polarity modes and multistage tandem mass spectrometric (MS(n)) measurements were performed on the ion trap analyser for positively charged tin-containing ions. The sum of complementary ions observed in the positive-ion mode (i.e. [M-R(3)](+) ion) and in the negative-ion mode (i.e. [R(3)](-) ion) permits molecular mass determination in spite of the fact that the molecular adducts were often missing even in the first-order mass spectra. The subsequent fragmentation of [M-R(3)](+) ions studied by MS(n) and the correlation of observed fragment ions with the expected structures of synthesized organotin(IV) compounds allowed us to understand the fragmentation behaviour and the mechanism of the ion formation for studied compounds. The typical neutral losses are alkenes, alcohols and aldehydes. The fragmentation pattern of one selected compound was supported by MS(n) measurements of an isotopically labelled analogue to confirm unusual ion-molecule reactions of some fragment ions with water in the ion trap.     

Analysis of xanthates by negative-ion fast atom bombardment mass spectrometry

A new technique using negative-ion fast atom bombardment mass spectrometry for the analysis of xanthates and related compounds is described. Electron impact and positive-ion fast atom bombardment mass spectrometry produced no structurally related fragment ions or observable molecular ions at the expected m/z values. It was demonstrated that negative-ion fast atom bombardment ionization was the most suitable method of ionization for structure elucidation studies for the compounds described.     

Rapid classification of White Oak (Quercus alba) and Northern Red Oak (Quercus rubra) by using pyrolysis direct analysis in real time (DART™) and time-of-flight mass spectrometry

Thirty-four samples of Red Oak (Quercus rubra) and fifty samples of White Oak (Quercus alba) were analyzed by pyrolytic direct analysis in real time (DART) ionization coupled with time-of-flight (TOF) mass spectrometry. Although significant differences were not observed in the positive-ion mass spectra, the negative-ion mass spectra showed clear differences. Principal component analysis (PCA) and linear discriminant analysis (LDA) were calculated for the relative abundances of 11 peaks in the negative-ion mass spectra including peaks tentatively assigned as representing deprotonated acetic, malic, gallic, dimethoxycinnamic, and ellagic acids. Leave one out cross validation (LOOCV) was 100% successful in classifying the samples for both PCA and LDA.     

252Cf-plasma desorption mass spectrometry of unmodified lipid A: fragmentation patterns and localization of fatty acids.

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.     

Mass spectral characteristics of okadaic acid and simple derivatives.

Electron ionization (EI), chemical ionization (CI) and fast-atom bombardment (FAB) mass spectra of the marine toxin okadaic acid and its synthetic methyl, pentafluorobenzyl, and trimethylsilyl ester and ether derivatives were generated. Several ionization conditions and ion-processing methods were used to obtain positive- and negative-ion conventional spectra and tandem (MS/MS) spectra. The EI and the positive-ion CI spectra provided fragment ions characteristic of the structure, and the negative-ion CI and FAB spectra provided molecular ions. The addition of alkali salts to the FAB matrix resulted in reduced fragmentation and the formation of intense alkali-metal-cationized molecules. Pentafluorobenzyl ester derivatives provided intense carboxylate ions under electron-capture ionization. Analytically useful MS/MS spectra were obtained by low-energy collision-induced decomposition of the carboxylate anion produced from the tetrasilylated pentafluorobenzylokadaate.     

3-Hydroxycoumarin as a new matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of DNA

3-Hydroxycoumarin (3-HC) was designed, synthesized, and tested as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of a variety of synthetic oligodeoxynucleotides ranging long from three to 70 bases. Using the matrix solution of 3-HC dissolved in a mixed solvent of acetone and diammonium hydrogen citrate, DNA segments over the mass range 800 Da to 6900 Da were isotopically resolved with high signal-to-noise (S/N) ratio. The individual isotopic molecular ion peaks of a group of 23-mer mixed-base oligomers differing by one or two bases with mass differences of 9 or 7 Da were recorded. Larger oligodeoxynucleotide segments of 34-mer, 50-mer, and 70-mer have also been analyzed effectively. Less than 250 attomol of a 10-mer DNA segment was clearly detected without any fragmentation. The new matrix can be used for the analysis of DNA segments in both positive- and negative-ion modes, and the quality of all negative-ion mode spectra are as good as that obtained in positive-ion mode shown in this paper. Compared with conventional matrices of 3-hydroxypicolinic acid (3-HPA) and 6-aza-2-thiothymine (ATT), 3-HC had noticeable improvement in resolution, S/N ratio, spot-to-spot-, and sample-to-sample reproducibility for analyzed DNA segments.     

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

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-ITMS(n)) in positive and negative ion modes. Both positive and negative ion mode MS(1)-MS(4) 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 MS(1)-MS(4) 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 MS(1)-MS(3) spectra was constructed for the identification of structural elements in the saponin. Peak intensity ratios in MS(3) 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.     

Analysis of amino acid mixtures by plasma desorption mass spectrometry

Plasma desorption mass spectrometry (PDMS) was investigated as a means of analysing mixtures of three, four and five amino acids in both positive- and negative-ion modes. Fifteen mixtures were tested; each mixture contained equimolar amounts of selected amino acids. The PD mass spectra exhibited MH⁺ and [M – H]^? molecular ions for all the aminoacids with different desorption–ionization yields. The spectra were more easily interpreted in the negative- than the positive-ion mode. The desorption order of the amino acids was progressively established by comparing the molecular ion desorption–ionization yields for each mixture. This desorption order was well correlated in both the positive- and negation-ion modes with the acid–base thermodynamic data for the amino acids in the gas phase. This observation gives some insight into the desorption–ionization mechanisms under PDMS conditions.     

Electrospray mass spectrometry and tandem mass spectrometry of bimetallic oxovanadium complexes

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

Direct structural assignment of neutral and sialylated N-glycans of glycopeptides using collision-induced dissociation MSn spectral matching

Mass spectrometric analyses of various N-glycans binding to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on collision-induced dissociation (CID) MS(n) spectra acquired by electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) in the positive- and negative-ion modes has been proposed as a direct method of assigning N-glycans without releasing them from N-glycopeptides. In the positive-ion mode of this approach, the MS(2) spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAcbeta1-4GlcNAc, GlcNAc: N-acetylglucosamine) attached to asparagine (N), and two charges on the [M+H+Na](2+) precursor ion are shared with both of the resulting fragments. These fragments are sodiated B(n)-type fragment ions of oligosaccharide (N-glycan) and a protonated peptide ion retaining one GlcNAc residue on the asparagine (N) residue. The structure of N-glycan was assigned by comparing MS(3) spectra derived from both the sodiated B(n)-type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MS(n) spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MS(n) spectra of deprotonated B(n)-type fragment ions of N-glycopeptide and the PA N-glycan standard. The efficacy of this approach was tested with chicken egg yolk glycopeptides with a neutral and a sialylated N-glycan, and human serum IgG glycopeptides with neutral N-glycan isomers. These results suggest that the approach based on MS(n) spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides.