Detection and structural characterization of amino polyaromatic hydrocarbon-deoxynucleoside adducts using fast atom bombardment and tandem mass spectrometry

Fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS) are shown to be useful methods for the detection and structural characterization of nanogram amounts of amino polyaromatic hydrocarbon-nucleoside DNA adducts. The positive ion spectra of four aromatic amine guanosine adducts were studied in detail. The FAB spectra of these adducts exhibit an [MH]+ ion and a more abundant aglycon fragment ion, [AH2]+, which results from the loss of the deoxyribose sugar. The sensitivity of the adducts to FAB was enhanced by preparing trimethylsilyl (TMS) ether derivatives. High-quality full-scan spectra could be obtained on less than 70 ng of the derivatized adducts without signal averaging. With a B/E-linked scan of the [MH]+ ion for the TMS2 species, these same adducts could be detected by examination of their metastable ion spectra at levels as low as 4-5 ng (S/N greater than 10). Collision-induced dissociation (CID) of the [MH]+ ion yields the aglycon fragment and an ion, S1, which results from cleavage through the sugar. The CID spectrum of the aglycon [AH2]+ ion is much more useful, providing structural information relating to the base, the polyaromatic hydrocarbon, and, possibly, the site of covalent attachment. Differentiation of isomeric aminophenanthrene-guanine adducts was demonstrated on the basis of the CID spectra of their respective [AH2]+ ions. The use of TMS derivatives also improves the sensitivity of these methods.     

De novo peptide sequencing using CID and HCD spectra pairs

In tandem mass spectrometry (MS/MS), there are several different fragmentation techniques possible, including, collision‐induced dissociation (CID) higher energy collisional dissociation (HCD), electron‐capture dissociation (ECD), and electron transfer dissociation (ETD). When using pairs of spectra for de novo peptide sequencing, the most popular methods are designed for CID (or HCD) and ECD (or ETD) spectra because of the complementarity between them. Less attention has been paid to the use of CID and HCD spectra pairs. In this study, a new de novo peptide sequencing method is proposed for these spectra pairs. This method includes a CID and HCD spectra merging criterion and a parent mass correction step, along with improvements to our previously proposed algorithm for sequencing merged spectra. Three pairs of spectral datasets were used to investigate and compare the performance of the proposed method with other existing methods designed for single spectrum (HCD or CID) sequencing. Experimental results showed that full‐length peptide sequencing accuracy was increased significantly by using spectra pairs in the proposed method, with the highest accuracy reaching 81.31%.     

Fast atom bombardment and tandem mass spectrometry for structure determination of cysteine, N-acetylcysteine, and glutathione adducts of xenobiotics

Fast atom bombardment mass spectrometry (FAB/MS) and FAB combined with tandem mass spectrometry (MS/MS) were examined for their applicability to the structure determination of xenobiotics conjugated with the members of the glutathione family (glutathione, cysteine, and N-acetylcysteine). Comparisons between FAB/MS and thermospray MS data are made. FAB/MS is generally successful at generating molecular ion species under both positive and negative ion conditions. Upon collisional activation the adducts undergo characteristic cleavages around the sulfur providing structural information about the conjugate. The analysis of the N-acetylcysteine conjugate isolated from rat urine is presented as an example of the application of FAB/MS/MS to biological problems.     

Characterization of cisplatin adducts of oligonucleotides by fast atom bombardment mass spectrometry

The products of the reaction of the antitumor drug cisplatin (cis-diamminedichloroplatinum(II)) with four oligonucleotide tetramers, d(GpCpGpC), d(GpGpCpC), d(TpGpApT), and d(TpGpCpT), were separated by gel permeation chromatography and characterized by negative- and positive-ion fast atom bombardment (FAB) mass spectrometry. Fragment ions indicating the oligonucleotide sequence and the position of cisplatin binding were observed in MS/MS spectra following collisional activation and B/E-linked scanning. Positive-ion FAB MS/MS spectra were characterized by platinum-containing product ions. Nonplatinated sequence ions and internal fragment ions were present primarily in the negative-ion spectra. The most prominent fragment ions containing platinum were [HB2.Pt.B3H]+ and [HB1.Pt.B2H]+, where B1, B2, and B3 were bases in the oligonucleotide tetramer, one of which was usually guanine. Both singly and doubly charged platinum complexes were observed, probably indicating reduction of Pt(II) during the FAB ionization process. The location of the platinum complex bound to each oligonucleotide sequence could be determined, and the binding sites observed by mass spectrometry were similar to those previously determined by other methods. FAB ionization with collisional activation and MS/MS analysis could serve as a new method for structural analysis of platinated oligonucleotides.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

High-performance liquid chromatography-mass spectrometry of glycosphingolipids: II. Application to neutral glycolipids and monosialogangliosides

We previously reported a method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of molecular species of GlcCer and IV3 beta Gal-Gb4Cer [M. Suzuki et al. (1989) J. Biochem. 105, 829-833]. In this paper, we report a modification of this HPLC/FAB/MS method, which was used for the separation and characterization of neutral glycosphingolipids (GlcCer, LacCer, Gb3Cer, Gb4Cer, and IV3 alpha GalNAc-Gb4Cer) and monosialogangliosides [GM3(NeuAc or NeuGc), GM2 (NeuAc or NeuGc), and GM1 (NeuAc or NeuGc)]. Mixtures of the purified neutral glycolipids and monosialogangliosides were subjected to HPLC on a silica gel column, with programmed elution with isopropanol-n-hexane-water, with or without ammonium hydroxide. In order to obtain mass spectra and mass chromatograms of individual components, effluent from the HPLC column was mixed with a methanol solution of triethanolamine, which was used as the matrix for the FAB ionization, and one-thirtieth of the effluent mixture was introduced into a mass spectrometer through a frit interface. A mixture of the five neutral glycolipids, 5 micrograms of each, gave five peaks on a mass chromatogram obtained by monitoring of the corresponding major pseudo-molecular ions. A mixture of the six monosialogangliosides, 5 micrograms of each, gave six peaks on a mass chromatogram obtained by monitoring of the major pseudo-molecular ions, indicating that GM3, GM2, and GM1 were clearly separated, and that separation due to differences in sialic acid species was also achieved. In the mass spectra of the neutral glycolipids and monosialogangliosides, pseudo-molecular ions and fragment ions due to the elimination of sugar moieties were clearly detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance liquid chromatography-mass spectrometry of glycosphingolipids: I. Structural characterization of molecular species of GlcCer and IV3 beta Gal-Gb4Cer

A method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of glycosphingolipids was developed, which involves a frit interface between the HPLC and the MS. The molecular species of glucosylceramide (GlcCer) purified from the spleen of a patient with Gaucher's disease and galactosylglobotetraosylceramide (IV3 beta Gal-Gb4Cer) from mouse kidney were analyzed using this system on a reversed-phase column, with methanol containing 1% glycerol as the elution solvent. The injection of 1 microgram of GlcCer gave the mass spectra of seven major molecular species, the pseudo-molecular ion for each of the seven molecular species being observed at m/z 698, 726, 754, 782, 808, 796, and 810, respectively. The injection of 200 pg of synthetic N-stearoyl glucosylsphingosine (d18:1) gave a clear peak with the single ion monitoring method detecting the pseudo-molecular ion at m/z 726. The injection of 5 micrograms of IV3 beta Gal-Gb4Cer gave the mass spectra of six major molecular species, the pseudo-molecular ions being observed at m/z 1,489, 1,471, 1,515, 1,497, 1,517, and 1,499. This report deals with a new HPLC/FAB/MS system, which was successfully applied to the structural characterization of the molecular species of neutral glycosphingolipids, and the system is a quite promising for development into a quantitative method for glycosphingolipids with high sensitivity and specificity.     

Profiling with structural elucidation of the neutral and anionic O-linked oligosaccharides in the egg jelly coat of Xenopus laevis by Fourier transform mass spectrometry

A strategic method with high speed and sensitivity is outlined for the analysis of mucin-type oligosaccharide from the jelly coat of Xenopus laevis. The method relies primarily on mass spectrometric techniques, in this case matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry (MALDI-FTMS) and collision-induced dissociation (CID). Separation with isolation of the oligosaccharides was streamlined to couple well with mass spectrometry allowing the rapid determination of all detectable components from both neutral and anionic species. Partial structures of anionic components, composed primarily of sulfate esters, were obtained with CID. For neutral species, a method that allowed the complete structural determination using mass spectrometry was used. The method builds on the structure of small number of known compounds to determine unknown structures from the same biological source. In this example, a small number of oligosaccharides, elucidated previously by NMR, were used to develop a set of substructural motifs that were characterized by CID. The presence of the motifs in the CID spectra were then used to determine the structures of unknown compounds that were in abundances too small for NMR analysis.     

Laccase-catalyzed polymerization of two phenolic compounds studied by matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with collision-induced dissociation experiments

Enzymatic oxidation of two phenolic compounds [syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) and 2,6-dimethylphenol] was studied. The products of laccase- and laccase-mediator-catalyzed oxidation reactions were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and further analyzed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) MS with collision-induced dissociation (CID) experiments. For the oligomers of syringic acid, some variability was observed in MALDI-TOF analysis. However, the origin of this variability could not be resolved on the basis of MALDI-TOF spectra due to the poor resolution of the instrument in use. The strength of ESI-FTICR MS was the high-resolution data provided from oligomers of syringic acid. The CID experiments were extremely useful for structural studies of oligomers and verified that the variability of the products was due to the end groups; the phenolic hydroxyl group was modified during the oxidation.     

Sequencing Regular and Labeled Oligonucleotides Using Enzymatic Digestion and Ionspray Mass Spectrometry

A method using a combination of enzymatic digestion and ionspray mass spectrometry (MS) was developed for sequencing oligodeoxynucleotides (ODNs) containing more than 20 bases. Phosphodiesterase (PDE) digestion of ODNs produced truncated ODNs whose molecular weights (MWs) were determined by ionspray MS. It was demonstrated that reconstruction of MW spectra over a large MW range produced easy-to-read sequence ladders similar to those obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF-MS). Sample and enzyme cleanup, digestion control, and MW reconstruction were found to be crucial factors. For regular ODNs, both 5′- and 3′-PDE digestions are needed for complete sequencing. Late in the time course of PDE digestions, 5′-nucleoside monophosphates were found to produce artifactual peaks in the reconstructed MW spectra, and a table correlating base compositions and MS ions was compiled to handle such situations. For labeled ODNs, it is necessary to use collision-induced dissociation–tandem mass spectrometry (CID–MS/MS) for complete sequence determination. Sequencing of regular 22-mer and labeled 18-mer ODNs was demonstrated in this work.     

pepgrep: A Tool for Peptide MS/MS Pattern Matching

Typically, detection of protein sequences in collision-induced dissociation (CID) tandem MS (MS2) dataset is performed by mapping identified peptide ions back to protein sequence by using the protein database search (PDS) engine. Finding a particular peptide sequence of interest in CID MS2 records very often requires manual evaluation of the spectrum, regardless of whether the peptide-associated MS2 scan is identified by PDS algorithm or not. We have developed a compact cross-platform database-free command-line utility, pepgrep, which helps to find an MS2 fingerprint for a selected peptide sequence by pattern-matching of modelled MS2 data using Peptide-to-MS2 scoring algorithm. pepgrep can incorporate dozens of mass offsets corresponding to a variety of post-translational modifications (PTMs) into the algorithm. Decoy peptide sequences are used with the tested peptide sequence to reduce false-positive results. The engine is capable of screening an MS2 data file at a high rate when using a cluster computing environment. The matched MS2 spectrum can be displayed by using built-in graphical application programming interface (API) or optionally recorded to file. Using this algorithm, we were able to find extra peptide sequences in studied CID spectra that were missed by PDS identification. Also we found pepgrep especially useful for examining a CID of small fractions of peptides resulting from, for example, affinity purification techniques. The peptide sequences in such samples are less likely to be positively identified by using routine protein-centric algorithm implemented in PDS. The software is freely available at http://bsproteomics.essex.ac.uk:8080/data/download/pepgrep-1.4.tgz.     

Primary structure of the hypertrehalosaemic factor II from the corpus cardiacum of the Indian stick insect, Carausius morosus, determined by fast atom bombardment mass spectrometry

The primary structure of hypertrehalosaemic factor II, isolated from the corpus cardiacum of the Indian Stick Insect Carausius morosus, has been assigned as Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 from its fast atom bombardment (FAB) mass spectrum and metastable scans of its FAB spectrum. The structure assigned shows close homology to other insect neuropeptides. A synthetic sample of this peptide gave the same FAB spectra, reversed-phase high-performance liquid chromatographic behavior, and biological behavior as the natural material. Mass spectrometric fragmentation of the synthetic peptide was examined by B/E linked scan and MIKES techniques in a two-sector mass spectrometer and by the MS/MS technique in a four-sector (tandem) spectrometer.     

Strategy for simulation of CID spectra of N-linked oligosaccharides toward glycomics

To develop a novel glycomics tool that can enable anyone to identify oligosaccharides very easily and quickly, we have recently constructed a library of observed multistage tandem mass (MS(n)) spectra for oligosaccharides. However, this approach requires the preparation of a large variety of structurally defined oligosaccharides. Therefore, simulation of the tandem mass spectrum for any given structure would be another powerful approach with which to improve the above method. By performing collision-induced dissociation (CID) experiments of sets of oligosaccharides complementarily labeled with (13)C(6)-D-galactose, we identified characteristic fragment patterns for each branch type of N-linked oligosaccharides. On the basis of these characteristic fragment patterns, we could simulate CID spectra for three isomeric oligosaccharides. In addition, we successfully demonstrated the identification of an oligosaccharide by matching its CID spectrum against the library of simulated tandem mass spectra. This strategy will be a useful tool for glycomics, as well as for approaches based on the library of observed MS(n) spectra.     

Evaluation of Chemical Derivatisation Methods for Protein Identification using MALDI MS/MS

In proteomic studies, assigning protein identity from organisms whose genomes are yet to be completely sequenced remains a challenging task. For these organisms, protein identification is typically based on cross species matching of amino acid sequence obtained from collision induced dissociation (CID) of peptides using mass spectrometry. The most direct approach of de novo sequencing is slow and often difficult, due to the complexity of the resultant CID spectra. For MALDI-MS, this problem has been addressed by using chemical derivatisation to direct peptide fragmentation, thereby simplifying CID spectra and facilitating de novo interpretation. In this study, milk whey proteins from the tammar wallaby (Macropus eugenii) were used to evaluate three chemical derivatisation methods compatible with MALDI MS/MS. These methods included (i) guanidination and sulfonation using chemically-assisted fragmentation (CAF), (ii) guanidination and sulfonation using 4-sulfophenyl isothiocyanate (SPITC) and (iii) derivatising the epsilon-amino group of lysine residues with Lys Tag 4H. Derivatisation with CAF and SPITC resulted in more protein identification than Lys Tag 4H. Sulfonation using SPITC was the preferred method due to the low cost per experiment, the reactivity with both lysine and arginine terminated peptides and the resultant simplified MS/MS spectra.*Australian Peptide Conference Issue.**This project was funded by an ARC Linkage grant to Deane supported by TGR Biosciences and facilitated by access to the Australian Proteome Analysis Facility established under the Australian Government’s Major National Research Facilities program.     

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.     

Physalaemin-like immunoreactive peptides from rabbit stomach

Two physalaemin (PHY)-like immunoreactive peptides, designated PHLIPs, have been purified from extracts of rabbit stomach tissue. Fast atom bombardment/mass spectrometry (FAB/MS) indicated that the m/z values for the PHLIP protonated molecular ions were 867.419 and 796.4. FAB/tandem MS spectra, coupled with a knowledge of the amino acid composition and the aid of a computerized fragment-matching program, indicated the amino acid sequences to be: (formula; see text) The sequences of PHLIPs-7 and -8 were confirmed with synthetic peptides. The PHY-antiserum cross-reactivity of the PHLIPs reflects homology at amino acid residues 1, 3, 4 and 5 for the mammalian and amphibian residues.     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.     

High-Sensitivity Mass Spectrometry for Analysis of Posttranslational Modifications

Pulsed fast atom bombardment ionization (pulsed-FAB) mass spectrometry has been developed to improve the sensitivity of tandem mass spectrometry (MS/MS), allowing it to be used for the analysis of very small samples. MS/MS, when used with a magnetic four-sector instrument coupled with the pulsed-FAB system, allows significant enhancement in product ion intensity of over ten-fold in magnitude over conventional FAB. MS/MS was applied to the structural analysis of a unique nuclear protein, designated p28, which was isolated from a histone fraction obtained from starfish testes. The results clearly show that protein p28 is a heterodimer composed of testicular histones H2B and H4 which are cross-linked between Gln⁹ of H2B and Lys⁵ of H4.     

FAB CID-MS/MS characterization of tetrasaccharide tri- and tetrasulfate derived from the antigenic determinant recognized by the anti-chondroitin sulfate monoclonal antibody MO-225

The fast atom bombardment (FAB) collision induced dissociation (CID)-mass spectrometry/mass spectrometry (MS/MS) technique was successfully applied to characterize and identify the structures of the immunoreactive trisulfated and tetrasulfated tetrasaccharides that were obtained from the chondroitin sulfate in a shark fin using a treatment with chondroitinase ABC.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MS/MS mass spectrometry/mass spectrometry - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose