Silver cluster interferences in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of nonpolar polymers.

Potential difficulties associated with background silver salt clusters during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of nonpolar polymers are reported. Silver salt cluster ions were observed from m/z 1500 to 7000 when acidic, polar matrices, such as 2,5-dihydroxybenzoic acid (DHB), all-trans-retinoic acid (RTA) or 2-(4-hydroxyphenylazo)benzoic acid (HABA), were used for the analysis of nonpolar polymers. These background signals could be greatly reduced or eliminated by the use of nonpolar matrices such as anthracene or pyrene. Representative examples of these background interferences are demonstrated during the analysis of low molecular weight nonpolar polymers including polybutadiene and polystyrene. Nonpolar polymers analyzed with acidic, polar matrices (e.g., RTA) and silver cationization reagents can yield lower quality mass spectral results when interferences due to silver clusters are present. Replacing the polar matrices with nonpolar matrices or the silver salts with copper salts substantially improved the quality of the analytical results. In addition, it was found that silver contamination cannot be completely removed from standard stainless steel sample plates, although the presence of silver contamination was greatly reduced after thorough cleaning of the sample plate with aluminum oxide grit. Carry-over silver may cationize polymer samples and complicate the interpretation of data obtained using nonpolar matrices in the absence of added cationization reagents.

Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry.

A new method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the direct analysis of the mass-silent post-transcriptionally modified nucleoside pseudouridine in nucleic acids has been developed. This method utilizes 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide to derivatize pseudouridine residues. After chemical derivatization all pseudouridine residues will contain a 252 Da 'mass tag' that allows the presence of pseudouridine to be identified using mass spectrometry. Pseudouridine residues can be identified in intact nucleic acids by obtaining a mass spectrum of the nucleic acid before and after derivatization. The mass difference (in units of 252 Da) will denote the number of pseudouridine residues present. To determine the sequence location of pseudouridine, a combination of enzymatic hydrolysis and mass spectrometric steps are used. Here, MALDI analysis of RNase T1 digestion products before and after modification are used to narrow the sequence location of pseudouridine to specific T1 fragments in the gene sequence. Further mass spectrometric monitoring of exonuclease digestion products from isolated T1 fragments is then used for exact sequence placement. This approach to pseudouridine identification is demonstrated using Escherichia coli tRNAS: This new method allows for the direct determination of pseudouridine in nucleic acids, can be used to identify modified pseudouridine residues and can be used with general modification mapping approaches to completely characterize the post-transcriptional modifications present in RNAs.

Gas-phase hydrogen/deuterium exchange of positively charged mononucleotides by use of Fourier-transform ion cyclotron resonance mass spectrometry.

The gas-phase structures of protonated (deoxy)nucleoside-5'- and 3'-monophosphates (mononucleotides) have been examined by the use of gas-phase hydrogen/deuterium (H/D) exchange and high-field Fourier-transform ion cyclotron resonance mass spectrometry. These nucleotides were reacted with three different deuterating reagents: ND3, D2O, and D2S, of which ND3 was the most effective. All mononucleotides fully exchanged their labile hydrogen for deuterium with ND3 with the exception of deoxycytidine-3'-monophosphate, deoxyadenosine-5'-monophosphate, adenosine-5'-monophosphate, and adenosine-3'-monophosphate. Semiempirical calculations demonstrate the presence of hydrogen bonding upon protonation of the purine mononucleotides which may lead to incomplete H/D exchange. H/D exchange rates differed between the deoxymononucleotides and the ribomononucleotides, suggesting that the 2'-OH group plays an important role in the exchange process. Reactions of nucleosides and mononucleotides with D2O demonstrate that a structure-specific long-lived ion-molecule complex between D2O and the mononucleotide involving the phosphate group is necessary for exchange to overcome the high-energy activation barrier. In contrast, a structure-specific long-lived ion-molecule complex between the mononucleotides and ND3 is not required for exchange to occur.

Interfacing a polymer-based micromachined device to a nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometer.

Here we report the design, fabrication, and operation of a polymer-based microchip device interfaced to a nanoelectrospray ionization source and a Fourier transform ion cyclotron resonance mass spectrometer. The poly(methyl methacrylate) micromachined device was fabricated using X-ray lithography to produce a network of channels with high aspect ratios. Fabrication of high aspect ratio channels allows for zero dead volume interfaces between the microchip platform and the nanoelectrospray capillary interface. The performance of this device was evaluated with standard peptide and protein samples. High-quality mass spectral data from peptide and proteins (and mixtures thereof) were obtained without any interfering chemical noise from the polymer or the developers and plasticizers used in the fabrication process. Sample cross-contamination is not a problem using this polymer-based microchip device as demonstrated by the sequential analysis of several proteins. The nanoelectrospray source was operated at flow rates from 20 to 100 nL/min using pressure-driven flow, and uninterrupted operation for several hours is demonstrated without any noticeable signal degradation. The ability to fabricate multiple devices using injection molding or hot-embossing techniques of polymers provides a lower cost alternative to silica-based devices currently utilized with mass spectrometry.

Analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

MALDI-MS is one of the most useful techniques available for determining biomolecule mass. It offers high mass accuracy, good sensitivity, simplicity, and speed. Because singly charged ions of oligonucleotides are typically observed, MALDI-MS spectra are easy to interpret. This unit presents protocols for sample preparation and purification, matrix preparation, and matrix/analyte sample preparation. It provides an introduction to the instrumentation and its calibration, and a discussion of some of the useful applications of MALDI-MS analysis in the study of oligonucleotides. This technique is typically used for 120-mer or smaller oligonucleotides.

Analysis of oligonucleotides by electrospray ionization mass spectrometry.

Because of the high molecular weights and thermal lability of biomolecules such as nucleic acids and protein, they can be difficult to analyze by mass spectrometry. Such analyses require a "soft" ionization method that is capable of generating intact molecular ions. In addition, most mass analyzers have a limited upper mass range that is not sufficient for studying these large molecules. ESI-MS can be used to analyze molecules with a molecular weight that is larger than the mass-to-charge ratio limit of the analyzer. This unit describes how ESI allows for analysis of high-molecular-weight compounds through the generation of multiply charged ions in the gas phase. It discusses analyzer configurations, solvent selection, and gives protocols for sample preparation. For applications of ESI-MS, the unit discusses molecular weight determination and gives protocols for sequencing and for analyzing oligonucleotide modifications.

A comparative study on methods of optimal sample preparation for the analysis of oligonucleotides by matrix-assisted laser desorption/ionization mass spectrometry.

Metal adducts (e.g., Na+ and K+) significantly hinder the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although a number of sample purification methods exist, to date no comparative study exists to determine the most efficient method for purifying oligonucleotides. The objective of this work was to perform such a study. Several different oligonucleotide samples were synthesized. Aliquots of these samples were then purposely contaminated with sodium acetate to generate representative contaminated (salted) oligonucleotide samples. A number of popular oligonucleotide purification techniques were then tested as to their effectiveness at removing Na+ from the salted samples. The effectiveness of Na+ removal was qualitatively assessed by comparing the MALDI mass spectra of the original sample, the salted sample, and the salted sample after purification. Micropipet tips packed with C18 reversed-phase packing material (e.g., Zip Tips) appear to be the most effective means of purifying the oligonucleotides investigated. Minidialysis was found to be an effective alternative for purifying higher molecular weight oligonucleotides (> 10,000 u).

Surfactant-aided, matrix-assisted laser desorption/ionization mass spectrometry of hydrophobic and hydrophilic peptides.

The analysis of hydrophobic and hydrophilic peptides in an aqueous medium using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. The key development allowing for simultaneous analysis of both hydrophobic and hydrophilic components of the sample mixture is the use of surfactants to solubilize the hydrophobic components in the MALDI matrix solution. A wide variety of anionic, cationic, zwitterionic, and nonionic surfactants were evaluated for their ability to assist in the generation of an abundant pseudomolecular ion from a model hydrophobic peptide ([tert-butoxycarbonyl]Glu[gamma-O-benzyl]-Ala-Leu-Ala[O-phenacyl ester]). The results indicate that the most successful surfactant among those studied for analyzing the model hydrophobic peptide is sodium dodecyl sulfate (SDS). SDS exhibited no interfering surfactant background ions, little to no loss of the acid-labile protecting groups from the model hydrophobic peptide, and an abundant pseudomolecular ion of the analyte. In addition, the use of surfactants is shown to be compatible with hydrophilic peptides as well. Mixtures of hydrophobic and hydrophilic peptides were characterized using surfactant-aided (SA) MALDI-MS, and it is demonstrated that all components are detectable once the surfactant is included in the sample solution. We conclude that the key benefit of using SA-MALDI-MS is its ability to simultaneously analyze hydrophobic and hydrophilic peptides from a single sample mixture, including synthetic peptides containing acid- and base-labile protecting groups.

Mononucleotide gas-phase proton affinities as determined by the kinetic method.

The goal of this work is to determine the proton affinities of (deoxy)nucleoside 5'- and 3'-monophosphates (mononucleotides) using the kinetic method with fast atom bombardment mass spectrometry. The proton affinities of the (deoxy)nucleoside 5'- and 3'-monophosphates yielded the following trend: (deoxy)adenosine monophosphates > (deoxy)guanosine monophosphates > (deoxy)cytidine monophosphates >> deoxythymidine/uridine monophosphates. In all cases the proton affinity decreases or remains the same with the addition of the phosphate group from those values reported for nucleosides. The proton affinity is dependent on the location of the phosphate backbone (5'-vs. 3'-phosphates): the 3'-monophosphates have lower proton affinities than the 5'-monophosphates except for the thymidine/uridine monophosphates where the trend is reversed. Molecular modeling was utilized to determine if multiple protonation sites and intramolecular hydrogen bond formation would influence the proton affinity measurements. Semiempirical calculations of the proton affinities at various locations on each mononucleotide were performed and compared to the experimental results. The possible influence of intramolecular hydrogen bonding between the nucleobases and the phosphate group on the measured and calculated proton affinities is discussed.

Polyamine co-matrices for matrix-assisted laser desorption/ionization mass spectrometry of oligonucleotides.

The analysis of oligonucleotides using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has led to the investigation of the use of matrix additives (i.e., co-matrices) to help improve the poor spectral quality commonly observed during the analysis of this class of compounds. The use of certain matrix additives in MALDI-MS has been investigated previously, and these additives have been shown to enhance the desorption/ionization efficiency of oligonucleotides during the MALDI experiment. Specifically, amine bases, such as piperidine, imidazole, and triethylamine, have been shown to improve mass spectral quality as assessed by improved molecular ion resolution and increased molecular ion abundance. These improvements occur due to competition between the oligonucleotide and the co-matrix for protons generated during the MALDI event. Co-matrices with proton affinities near or above the proton affinities of the nucleotide residues serve as proton sinks during the desorption/ionization process. In this work, we have investigated the use of polyamines as co-matrices for MALDI mass spectrometric analysis of oligonucleotides. Spermine tetrahydrochloride, spermine, spermidine trihydrochloride, and spermidine were evaluated for their effectiveness at enhancing the mass spectral quality of oligonucleotides analyzed using MALDI-MS. The solution-phase pK( b) values and the gas-phase proton affinities of these polyamines were determined, and it was found that the polyamines appear to be more basic than the monofunctional amines investigated previously. The mass spectral data shows that spermidine and spermine are extremely effective co-matrices, yielding improved molecular ion resolution and molecular ion abundances. The spermine co-matrices are more effective than the spermidine co-matrices, but adduction problems with the spermine co-matrices limits their overall utility. In general, polyamine co-matrices are found to be more effective than monofunctional amine co-matrices at improving the mass spectral data obtained during MALDI-MS of oligonucleotides.

Electrospray ionization mass spectrometry of metalloporphyrins.

The magnesium, nickel, copper, zinc and vanadium metalloporphyrins from octaethylporphyrin, etioporphyrin I and tetraphenylporphyrin were characterized using electrospray ionization mass spectrometry (ESI-MS). The ion abundance of each of the porphyrins present in binary mixtures was monitored as a function of the porphyrin concentration and is dependent on the metalloporphyrin oxidation potential. It was found that, for binary mixtures of metalloporphyrins whose oxidation potentials differ by less than 0.1 V, the resulting ion abundance of each species is directly proportional to the concentration of each analyte in the mixture. For binary mixtures whose oxidation potentials differ by more than 0.1 V, relative abundances of the radical cations of each metalloporphyrin are determined by the oxidation potential and concentration of each metalloporphyrin with the analyte of lowest oxidation potential being ionized preferentially. The ability to ionize selectively one porphyrin over another in a binary mixture offers the potential to use ESI-MS for the qualitative analysis of porphyrins present in complex mixtures.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the analysis of RNase H cleavage products.

RNase H is an endonuclease which cleaves RNA at points of hybridization with DNA. However, certain ambiguities exist in terms of its specificity and location of cleavage along the RNA strand. The analysis of RNase H reaction products of an oligoribonucleotide hairpin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is demonstrated. The oligoribonucleotide studied has a highly stable secondary structure which reduces the efficiency of hybridization with the chimeric oligonucleotide used to direct RNase H cleavage. By monitoring the reaction products under different conditions using MALDI/TOF-MS, the optimum variables for cleavage of this highly stable hairpin structure can be determined.

Indirect mass spectrometric methods for characterizing and sequencing oligonucleotides.

The use of mass spectrometry for the characterization and sequence determination of oligonucleotides is reviewed. This review focuses primarily on the use of mass spectrometry to analyze sequence-specific fragments of oligonucleotides that are generated via solution-phase chemical reactions. The majority of these "indirect" sequencing methods are a result of recent advances in electrospray ionization and matrix-assisted laser desorption/ionization for the generation of intact gas-phase ions from oligonucleotides. Descriptions of the current indirect sequencing protocols will be presented as well as a comparison of the applicability of these procedures for analyzing "real world" samples. The applicability of indirect mass spectrometric sequencing to antisense oligonucleotides will be discussed in detail. © 1997 John Wiley & Sons, Inc.

An anticodon sequence mutant of Escherichia coli initiator tRNA: possible importance of a newly acquired base modification next to the anticodon on its activity in initiation.

Initiator tRNAs from eubacteria and chloroplasts lack a base modification next to the anticodon. This is in contrast to virtually all other tRNAs from these sources. We show that a mutant Escherichia coli initiator tRNA which has an anticodon sequence change from CAU to CUA now has a 2-methylthio-N6-(delta 2-isopentenyl)adenosine (ms2i6A) modification, produced by posttranscriptional modification of A, next to the anticodon. This newly acquired base modification may be important for the function of the mutant tRNA in initiation. In a miaA mutant strain of E. coli defective in biosynthesis of ms2i6A, the mutant initiator tRNA is 10- to 12-fold less active in initiation. The mutant tRNA is aminoacylated and formylated normally in the miaA strain. Thus, the absence of the base modification affects the activity of the mutant tRNA at a step subsequent to its formylation.

Characterization of oligonucleotides and nucleic acids by mass spectrometry.

The continued refinement of two recent methods for producing gas-phase ions, electrospray ionization and matrix-assisted laser desorption ionization, has resulted in new techniques for the rapid characterization of oligonucleotides by mass spectrometry. Using commercially available instruments, molecular mass measurements at the 20-mer level, with errors less than 2 Da, can now be made routinely in less than 15 min. Progress has also been achieved in the development of mass spectrometry for rapid sequencing of oligonucleotides smaller than 25 residues.

Ion trajectories in an electrostatic ion guide for external ion source fourier transform ion cyclotron resonance mass spectrometry.

An electrostatic ion guide (EIG) that consists of concentric cylinder and central wire electrodes can transport ions efficiently from an external ion source to an ion cyclotron resonance (ICR) ion trap for mass analysis, with several advantages over current injection methods. Because the electrostatic force of the EIG captures ions in a stable orbit about the wire electrode, ions with initially divergent trajectories may be redirected toward the ICR ion trap for improved ion transmission efficiency. SIMION trajectory calculations (ion kinetic energy, 1-200 eV; elevation angle, 0.30 °; azimuthal angle, 0.360°) predict that ions of m/z 1000 may be transmitted through a strong (0.01 → 3.0-T) magnetic field gradient. Judicious choice of ion source position and EIG potential minimizes the spread in ion axial kinetic energy at the ICR ion trap. Advantages of the EIG include large acceptance angle, even for ions that have large initial kinetic energy and large radial displacement with respect to the central z-axis, low ion extraction voltage (5-20 V), and efficient trapping because ions need not be accelerated to high velocity to pass through the magnetic field gradient.

Molecular mass measurement of intact ribonucleic acids via electrospray ionization quadrupole mass spectrometry.

The use of electrospray ionization mass spectrometry for the accurate determination of molecular masses of polynucleotides and small nucleic acids is developed. The common problem of gas phase cation adduction that is particularly prevalent in the mass spectrometric analysis of nucleic acids is reduced through the use of ammonium acetate precipitations and by the addition of chemical additives that compete for adduct ions in solution. The addition of chelating agents such as trans-1,2-diaminocyclohexane-N,N,N,',N'-tetraacetic acid to remove divalent metal ions and triethylamine to displace monovalent cations from the analyte, in conjunction with ammonium acetate precipitation, reduces cation adduction to levels that permit accurate mass analysis (mass errors of less than 0.01%) without further complex cleanup procedures. The potential utility of accurate mass measurements of small ribonucleic acids is discussed.

Summary: the modified nucleosides of RNA.

A comprehensive listing is made of posttranscriptionally modified nucleosides from RNA reported in the literature through mid-1994. Included are chemical structures, common names, symbols, Chemical Abstracts registry numbers (for ribonucleoside and corresponding base), Chemical Abstracts Index Name, phylogenetic sources, and initial literature citations for structural characterization or occurrence, and for chemical synthesis. The listing is categorized by type of RNA: tRNA, rRNA, mRNA, snRNA, and other RNAs. A total of 93 different modified nucleosides have been reported in RNA, with the largest number and greatest structural diversity in tRNA, 79; and 28 in rRNA, 12 in mRNA, 11 in snRNA and 3 in other small RNAs.