Fast atom Bombardment Mass Spectrometry of Nucleosides,Nucleotides and Oligonucleotides

Abstract

Fast atom bombardment (FAB) mass spectrometry, a new ionization technique, has been applied to a variety of polar, nonvolatile compounds with considerable success. Current literature regarding the analysis of nucleosides, nucleotides and oligonucleotides using FAB is reviewed.     

Desorption Chemical Ionization Mass Spectra of Underivatized Abscisic Acid-conjugated Metabolites,a Useful Aid for Structural Characterization

The use of desorption chemical ionization (D/CI) mass spectrometry for the structural characterization of the abscisic acid (ABA)-conjugated metabolites, abscisic acid-γ-d-glucosyl ester (ABAGE), 4′-O-dihydrophaseic acid-γ-d-glucoside (DPAGS) and γ-hydroxy-γ-methylglutaryl-hydroxyabscisic acid (HMG-HOABA), was studied. The effects of the D/CI source temperature on the spectral features are described. Enhanced quasi-molecular ion intensities (e.g., [M·NH₄]⁺) were observed in the D/CI(NH₃) spectrum at a low source temperature (170°C). When a higher source temperature (250°C) was used, more extensive fragmentation occurred, and structurally diagnostic fragment ions appeared in the spectra. The spectra obtained at two degrees of source temperature were complementary to each other for the structural characterization.     

Mass Spectrometry: Applications in the Analysis of Nucleosides,Nucleotides and Oligonucleotides

Abstract

The past ten years have been an exciting time in mass spectrometry as a number of important instrumental developments have revolutionized the field, including the analysis of nucleic acid components.^1,2 The focus of this talk will be on the impact that new ionization methods, e.g., plasma desorption(PD) and fast atom bombardment(FAB), and new magnet technology (expanded mass range and scan speed capability) have had on the analysis of nucleosides and nucleotides. Results from the speaker's laboratory will be used to illustrate the significance of capillary GC/MS techniques for the separation and analysis of complex mixtures of nucleosides derived from a biological source. In addition, some approaches being developed to overcome current limitations in the FAB analysis of nucleosides and nucleotides will be described. Unfortunately, time does not permit a discussion of other new areas of interest, i.e., LC/MS³ and MS/MS.⁴     

The electron impact, chemical ionization and fast atom bombardment positive ion mass spectra of 1,2-bis(sulfonyl)methylhydrazines.

A series of bis(sulfonyl)-1-methylhydrazines were analyzed by positive ion electron impact (EI), chemical ionization (CI) and fast atom bombardment (FAB) mass spectrometry. Since these compounds showed activity against the L1210 leukemia, an understanding of their mass spectral behavior is important should the structural characterization of metabolites be required. FAB proved to be the most useful technique, generally providing abundant protonated molecule ion peaks, in contrast to the weak peaks observed with CI (ammonia or isobutane) and the total absence of molecular ion peaks in the EI mass spectra. In addition, utilizing FAB eliminated the problem of thermal decomposition, which was very difficult to control under EI and CI experimental conditions. Fragments observed in FAB and CI mass spectra were consistent with protonation at the methyl-bearing nitrogen. One can locate the R1 and R2 moieties relative to the methyl-bearing nitrogen in FAB and CI by assigning that nitrogen as the site of protonation, with subsequent elimination of R2SO2H.     

Computerized Interpretation of the Electron Ionization Mass Spectra of Nucleoside TMS Derivatives

Abstract

A computer program has been developed for the automated interpretation of mass spectra of TMS derivatives of nucleosides found in human urine. The m/z values in the unknown spectrum are compared to m/z values of 3 different ion series commonly observed in the mass spectra of nucleoside TMS derivatives.¹ If a correlation exists, the unknown spectra are marked with color according to the scheme: 1) blue—molecular ion series, 2) red—base ion series and 3) yellow—sugar ion series. The program suggests a structural assignment for each of the marked ions and calculates a series related ion current. The calculated ion current is used to assign the of sugar contained in the unknown nucleoside.     

Peptide Identification from Mixture Tandem Mass Spectra

The success of high-throughput proteomics hinges on the ability of computational methods to identify peptides from tandem mass spectra (MS/MS). However, a common limitation of most peptide identification approaches is the nearly ubiquitous assumption that each MS/MS spectrum is generated from a single peptide. We propose a new computational approach for the identification of mixture spectra generated from more than one peptide. Capitalizing on the growing availability of large libraries of single-peptide spectra (spectral libraries), our quantitative approach is able to identify up to 98% of all mixture spectra from equally abundant peptides and automatically adjust to varying abundance ratios of up to 10:1. Furthermore, we show how theoretical bounds on spectral similarity avoid the need to compare each experimental spectrum against all possible combinations of candidate peptides (achieving speedups of over five orders of magnitude) and demonstrate that mixture-spectra can be identified in a matter of seconds against proteome-scale spectral libraries. Although our approach was developed for and is demonstrated on peptide spectra, we argue that the generality of the methods allows for their direct application to other types of spectral libraries and mixture spectra.The success of tandem MS (MS/MS¹) approaches to peptide identification is partly due to advances in computational techniques allowing for the reliable interpretation of MS/MS spectra. Mainstream computational techniques mainly fall into two categories: database search approaches that score each spectrum against peptides in a sequence database (1–4) or de novo techniques that directly reconstruct the peptide sequence from each spectrum (5–8). The combination of these methods with advances in high-throughput MS/MS have promoted the accelerated growth of spectral libraries, collections of peptide MS/MS spectra the identification of which were validated by accepted statistical methods (9, 10) and often also manually confirmed by mass spectrometry experts. The similar concept of spectral archives was also recently proposed to denote spectral libraries including “interesting” nonidentified spectra (11) (i.e. recurring spectra with good de novo reconstructions but no database match). The growing availability of these large collections of MS/MS spectra has reignited the development of alternative peptide identification approaches based on spectral matching (12–14) and alignment (15–17) algorithms.However, mainstream approaches were developed under the (often unstated) assumption that each MS/MS spectrum is generated from a single peptide. Although chromatographic procedures greatly contribute to making this a reasonable assumption, there are several situations where it is difficult or even impossible to separate pairs of peptides. Examples include certain permutations of the peptide sequence or post-translational modifications (see (18) for examples of co-eluting histone modification variants). In addition, innovative experimental setups have demonstrated the potential for increased throughput in peptide identification using mixture spectra; examples include data-independent acquisition (19) ion-mobility MS (20), and MS^E strategies (21).To alleviate the algorithmic bottleneck in such scenarios, we describe a computational approach, M-SPLIT (mixture-spectrum partitioning using library of identified tandem mass spectra), that is able to reliably and efficiently identify peptides from mixture spectra, which are generated from a pair of peptides. In brief, a mixture spectrum is modeled as linear combination of two single-peptide spectra, and peptide identification is done by searching against a spectral library. We show that efficient filtration and accurate branch-and-bound strategies can be used to avoid the huge computational cost of searching all possible pairs. Thus equipped, our approach is able to identify the correct matches by considering only a minuscule fraction of all possible matches. Beyond potentially enhancing the identification capabilities of current MS/MS acquisition setups, we argue that the availability of methods to reliably identify MS/MS spectra from mixtures of peptides could enable the collection of MS/MS data using accelerated chromatography setups to obtain the same or better peptide identification results in a fraction of the experimental time currently required for exhaustive peptide separation.     

Differentiation of Isomeric Purine and Pyrimidine Mononucleotides by Fast Atom Bombardment Tandem Mass Spectrometry

Abstract

The use of positive ion fast atom bombardment mass-analysed ion kinetic energy (FAB/MIKE) spectroscopy to differentiate the 2′, 3′-and 5′-monophosphate isomers of adenosine, guanosine and cytidine is described.     

Enhancement of structural information in FAB ionized carbohydrate samples by neutral gas collision

Fast atom bombardment (FAB) ionization and two coupled analyzers (BE-EB) have been combined with neutral gas collision (C) to enhance structural information in the mass spectra of oligosaccharides. (B and E are abbreviations for magnetic and electric sectors respectively.) FAB ionization and the first analysers (BE) have provided parent ions free from biological and liquid matrix contaminants. Structural detail of these products were observed after collision and daughter ion analysis in a second coupled analyser (EB). Starting from complex mixtures, this instrumental approach, BE-C-EB, has provided specific oligomeric sequence information which was not observed in the normal FAB mass spectra. Collision spectra obtained from isomeric linear and branched oligosaccharides show unique fragments that can be directly related to structure.     

7-Carbapurine Ribofuranosides: Synthesis by Solid-Liquid Phase-Transfer Glycosylation and 15N-NMR Spectra

Abstract

The synthesis of pyrrolo [2,3-d]pyrimidine ribofuranosides by solid-liquid phase-transfer glycosylation is described and ¹⁵N NMR spectra of 7-carbapurine nucleosides are reported.     

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.     

Mass spectrometric localization of carbon-carbon double bonds: A critical review of recent methods

Current methods are reviewed for determining the position of double bonds in fatty acids, and other unsaturated organic compounds, using mass spectrometry. ‘On-site’ and ‘remote-site’ derivatization methods are described, and their advantages and disadvantages for mass spectrometric analysis discussed. Chemical transformation of double bonds by methoxylation, silyloxylation or deuteration, together with electron impact (EI), chemical ionization (CI) or collisionally induced decomposition (CID) techniques in combination with fast atom bombardment (FAB) or CI, are found to be most suitable for polyunsaturated fatty acids (PUFA): for the analysis of less unsaturated compounds on a submicrogram scale those methods are most promising which either do not involve derivatization of the double bonds, or give derivatives in quantitative yields. Effects of mass spectrometer geometry and operating conditions are also considered.     

An Unsupervised, Model-Free, Machine-Learning Combiner for Peptide Identifications from Tandem Mass Spectra

As the speed of mass spectrometers, sophistication of sample fractionation, and complexity of experimental designs increase, the volume of tandem mass spectra requiring reliable automated analysis continues to grow. Software tools that quickly, effectively, and robustly determine the peptide associated with each spectrum with high confidence are sorely needed. Currently available tools that postprocess the output of sequence-database search engines use three techniques to distinguish the correct peptide identifications from the incorrect: statistical significance re-estimation, supervised machine learning scoring and prediction, and combining or merging of search engine results. We present a unifying framework that encompasses each of these techniques in a single model-free machine-learning framework that can be trained in an unsupervised manner. The predictor is trained on the fly for each new set of search results without user intervention, making it robust for different instruments, search engines, and search engine parameters. We demonstrate the performance of the technique using mixtures of known proteins and by using shuffled databases to estimate false discovery rates, from data acquired on three different instruments with two different ionization technologies. We show that this approach outperforms machine-learning techniques applied to a single search engine’s output, and demonstrate that combining search engine results provides additional benefit. We show that the performance of the commercial Mascot tool can be bested by the machine-learning combination of two open-source tools X!Tandem and OMSSA, but that the use of all three search engines boosts performance further still. The Peptide identification Arbiter by Machine Learning (PepArML) unsupervised, model-free, combining framework can be easily extended to support an arbitrary number of additional searches, search engines, or specialized peptide–spectrum match metrics for each spectrum data set. PepArML is open-source and is available from . Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Application of fast atom bombardment mass spectrometry to biological samples: analysis of urinary metabolites of acetaminophen

Fast atom bombardment (FAB) is useful for the characterization of all major metabolites of the analgesic acetaminophen (APAP). It is particularly useful for providing mass spectra of the polar glucuronide and sulfate conjugates which eluded identification by field desorption and other more conventional methods of ionization. A protocol is described for the use of FAB in the identification of urinary APAP metabolites isolated by reversed phase high-performance liquid chromatography (HPLC) following therapeutic dosages of the drug. A tentative set of recommendations for the off-line use of HPLC and FAB is directed towards solving problems encountered when using these two analytical techniques in concert. In addition, a method for calculating the signal to background ratio (S/B) for analyte peaks in FAB spectra from selected relative ion intensities is proposed. Examples are presented that show the potential of S/B as an empirical parameter for judging the quality of FAB spectra.     

Mass spectrometry of sterols. Electron ionization induced fragmentation of C-4-alkylated cholesterols

The electron impact ionization of C-4-alkylated cholest-5-en-3β-hydroxysterols has been investigated. The mass spectra of the C-4-alkylated cholesterols contain a number of ions in the high mass region for which analogous ions are not found in the spectrum of cholesterol. Detailed studies of the composition and origin of these ions have been made by high resolution mass spectrometry and analysis of metastable ions. In addition, a large number of isotopically (deuterium and ¹⁸O) substituted C-4-alkylated analogues have been prepared to assist in the interpretation of the spectra. The combined results indicate the occurrence of a number of very complex and unusual electron ionization induced fragmentations. Most notable of the findings reported herein concerns the demonstration of the formation of an ion involving loss of the elements of ring A with an intramolecular shift of the oxygen and hydrogen atoms of the hydroxyl function to the charge-retaining species.     

Isolation and identification of 1(3),2-diacylglyceryl-(3)-O-4'-(N,N,N-trimethyl)homoserine from the soil amoeba, Acanthamoeba castellanii

A polar lipid accounting for 12.5% of the total lipid nitrogen has been isolated from the protozoan Acanthamoeba castellanii. On the basis of thin-layer chromatography and mass spectral analysis, the lipid has been identified as diacylglyceryltrimethylhomoserine (DGTS). Fast atom bombardment (FAB) mass spectra of DGTS are reported for the first time and are compared to the FAB mass spectra of phosphatidylcholines and the electron ionization (EI) and field desorption (FD) mass spectra of DGTS. Gas-liquid chromatographic-mass spectrometric (GLC-MS) analysis of the acyl chain composition of this lipid has shown that 87.5% consists of cis-9-octadecenoic acid. Plasma membrane isolated from this organism has shown that labeled DGTS appears in the plasma membrane but is not enriched in this fraction. DGTS has been isolated previously only from a limited number of green plants and one species of fungus. Identification of this lipid in Acanthamoeba indicates that this lipid is distributed among a diverse group of lower eucaryotes.     

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.     

Spectral analysis of a series of partially protected and deprotected tetrapeptides, analogues of AS-I phytotoxin

Summary A series of six tetrapeptides, analogues of AS-I phytotoxin, pathogenic to sunflower, have been synthesized either in solution and/or by solid phase methods and have been tested for phytotoxic activity in various plants and cytotoxic activity in three cancer cell lines. These peptides were identified as model compounds by fast atom bombardment (FAB), plasma desorption (PD), electrospray ionization (ESI) mass spectrometry and by¹H,¹H-¹H,¹³C and¹H-¹³C NMR. The data presented show that in protected tetrapeptides the molecular ion was easily identified whereas some difficulties appeared with the fully deprotected peptides. NMR spectra are given.     

Distinction between α- and γ-Glutamyl Dipeptides by Means of NMR Spectrometer and Amino Acid Analyzer

Conjugated metabolites of abscisic acid (ABA) have been characterized by their chemical ionization (CI) mass spectra using isobutane and ammonia as reagent gases. The CI mass spectra usually consist of quasimolecular ions ([QM]⁺), and constituent aglycone- and.sugar-derived ions. Hence, the spectra are simply interpreted and afford useful information for structutal characterization. CI examinations of ABA and its unconjugated metabolites and collisionally activated dissociation (CAD) measurements of selected ions were helpful in characterizing the constituent aglycones. The distinction between the two glycosidal forms, glycoside and glycosyl ester, has been also discussed in the corresponding conjugates. Confirmation of the fragmentation pathways via recognition of the diagnostic ions can be made by the extensive use of ammonia- d₃ as an additional reagent gas.     

Time-of-flight Secondary Ion Mass Spectrometry of Branched RNA Fragrants: Messenger RNA Splicing Intermedistes

Abstract

The negative ion mass spectra generated by a reflecting time-of-flight mass spectrometer are reported for a series of protected oligonucleotides. Quasimolecular and sequence ions have been detected, and the location and nature of protecting groups have been confirmed.     

MALDI AND FAB MASS SPECTROMETRY OF NUCLEOSIDE TRIPHOSPHATES: A COMPARATIVE STUDY

Abstract

Comparison of MALDI and FAB mass spectra of dATP, dTTP, dCTP, and dGTP shows that the former technique gives clear molecular ions with minimal fragmentation whereas the latter gives more fragment ions and weaker parent peaks.