Multi-charged oligonucleotide ion formation in sonic spray ionization.

An oligonucleotide tends to release hydrogen atoms from a phosphoric acid group and to form negative ions that can be detected by mass spectrometry. Usually, with a solution-spray based ionization technique, the negative ions are present in different charge states. Ion formation for the nucleotide is quite complicated and is easily influenced by matrix and other constituents in a sample solution, as well as by the operating parameters for a mass spectrometer. In this work, we studied oligonucleotide ion formation by using an ion trap mass spectrometer combined with a sonic spray ionization (SSI) source. An oligonucleotide with 20 bases was measured. Effects from contaminants and parameters affecting the ion production, such as a high voltage applied to the ionization source and sample solution-flow rate, were investigated. Our results showed that an ion with about one charge for every three bases was most abundant. However, the signal intensity and the mass spectrum pattern were sensitive to the matrix and operating parameters. One of the reasons for such sensitivity is that there are various ion states for an oligonucleotide. Any change in the matrix or an operating parameter may shift the balances between the ion states. Adding Tris, or (hydroxymethyl)aminomethane, enhanced the signal intensity of the oligonucleotide and promoted formation of the oligonucleotide ion with higher charges, while adding acetic acid favored the ions with lower charges, compared with that obtained in the medium without adding Tris and acetic acid. The effects on charged droplets and chemical enhancement were investigated. The mechanism for oligonucleotide ion formation is discussed.     

Measuring salty samples without adducts with MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been used for the discovery of hundreds of novel cell to cell signaling peptides. Beyond its advantages of sensitivity and minimal sample preparation requirements, MALDI MS is attractive for biological analyses as high quality mass spectra may be obtained directly from specific locations within prepared tissue sections. However, due to the large quantity of salts present in physiological tissues, these mass spectra often contain many adducts of cationic salts such as sodium and potassium, in addition to the molecular ion [M + H]⁺. To reduce the presence of cation adducts in MALDI mass spectra obtained directly from tissues, we present a methodology that uses a slow condensation procedure to enable the formation of distinct regions of matrix/analyte crystals and cation (salt) crystals. Secondary ion mass spectrometric imaging suggests that the salts and MALDI matrix undergo a mutually exclusive crystallization process that results in the separation of the salts and matrix in the sample.     

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).     

Evading metal adduct formation during desorption‐ionization mass spectrometry

An investigation into the propensity of metal adduct formation in the recently developed Desorption Ionization by Charge Exchange (DICE) mass spectrometric technique has demonstrated that this method could be utilized to minimize spectral complications caused by metal adducts. For example, peaks for sodium and other metal adducts were not observed in the mass spectra acquired by the ambient‐pressure DICE technique from samples deposited on a solid surface, even after the salt content of samples was deliberately increased. A mass spectrum recorded from a urine sample by this technique showed peaks only for the proton adducts of urea and creatinine. This technique employs a nebulized spray of charged toluene droplets for analyte desorption. Because of the non‐polar nature of the spray reagent, it neither contains any appreciable amount of cations nor provides any favored ‘pickup’ of metal cations from the sample matrix. Consequently, peaks for metal adducts that are commonly observed with other desorption techniques are minimal or absent in the spectra recorded by the DICE method. Copyright © 2010 John Wiley & Sons, Ltd.     

Oligonucleotides with 2'-O-carboxymethyl group: synthesis and 2'-conjugation via amide bond formation on solid phase

An efficient method for synthesis of oligonucleotide 2'-conjugates via amide bond formation on solid phase is described. Protected oligonucleotides containing a 2'-O-carboxymethyl group were obtained by use of a novel uridine 3'phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group is stable to the conditions used in solid-phase oligonucleotide assembly, but easily removed by Pd(0) and morpholine treatment. 2'-O-Carboxymethylated oligonucleotides were then efficiently conjugated on a solid support under normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high purity in good yield. The method is well suited in principle for the preparation of peptide-oligonucleotide conjugates containing an amide linkage between the 2'-position of an oligonucleotide and the N-terminus of a peptide.     

Matrix effect on the analysis of oligonucleotides by using a mass spectrometer with a sonic spray ionization source.

Matrix or impurities remaining in a DNA sample solution after various sample treatment procedures may influence a subsequent DNA analysis. In this work, several matrices were investigated concerning their effects on the analysis of oligonucleotide by using an ion-trap mass spectrometer equipped with a sonic spray ionization source. Inorganic salts of sodium chloride and magnesium chloride depressed the signal intensity by about 50% when the content of the salts was about 10 microM. dNTPs and Taq showed more severe depression on the oligonucleotide. However, Tris, or (hydroxymethyl)aminomethane, intensified the signal intensity, if its content was within an appropriate range. When the content of Tris was about 500 microM, the signal intensity was enhanced by factors of 3 and 5 for the 6-mer and the 20-mer oligonucleotides, respectively. With the existence of Tris, matrix effects from the inorganic salts, dNTPs and Taq were reduced.     

DNA-templated Ag nanocluster formation

The high affinity of Ag+ for DNA bases has enabled creation of short oligonucleotide-encapsulated Ag nanoclusters without formation of large nanoparticles. Time-dependent formation of cluster sizes ranging from Ag1 to Ag4/oligonucleotide were observed with strong, characteristic electronic transitions between 400 and 600 nm. The slow nanocluster formation kinetics enables observation of specific aqueous nanocluster absorptions that evolve over a period of 12 h. Induced circular dichroism bands confirm that the nanoclusters are associated with the chiral ss-DNA template. Fluorescence, absorption, mass, and NMR spectra all indicate that multiple species are present, but that their creation is both nucleotide- and time-dependent.     

Role of the preparation procedure in the formation of spherical and monodisperse surfactant/polyelectrolyte complexes

Complexes formed by a double-tail cationic surfactant, didodecyldimethyl ammonium bromide, and an anionic polyelectrolyte, an alternating copolymer of poly(styrene-alt-maleic acid) in its sodium salt form, were investigated with respect to variation in the charge ratio (x) between the polyelectrolyte negative charges and the surfactant positive charges. The morphology and microstructure of the complexes were studied by light microscopy and small-angle X-ray scattering for different preparation conditions. Independent of the sample preparation procedure and the charge ratio x, the X-ray results show that the microscopic structure of the complexes is a condensed lamellar phase. By contrast, the morphology of the complexes changes dramatically with the preparation procedure. The complexes formed by mixing a surfactant solution and a polyelectrolyte solution strongly depend on x and are always extremely heterogeneous in size and shape. Surprisingly, we show that, when the two solutions interdiffuse slowly, spherical complexes of micrometric and rather uniform size are systematically obtained, independently on the initial relative amount of surfactant and polyelectrolyte. The mechanism for the formation of these peculiar complexes is discussed.     

Two-peak phenomena and formation origin in micellar electrokinetic capillary chromatography

Since Terabe et al.[1] developed micellar electrokinetic capillary chromatography (MECC) in 1984, a great number of important advances about separating neutral compounds have been achieved. In MECC mode, micellar of an ionic surfactant can form so-called pseudo stationary phase in the buffer solution when it is above the critical micelle concentration, and some portions of the solute may be distributed into the micellar phase when they are mobilized into the buffer solution from sample zone…     

Using sol-gel/crown ether hybrid materials as desalting substrates for matrix-assisted laser desorption/ionization analysis of oligonucleotides

This study demonstrates the feasibility of using sol-gel/crown ether hybrid materials as sample substrates that reduce the intensity of the signals of sodium ion adducts of oligonucleotides during matrix-assisted laser desorption/ionization (MALDI) analysis. 2-Hydroxymethyl[15]crown-5 and 2-hydroxymethyl[18]crown-6 were added as dopants during the sol-gel process to generate desalting substrates for MALDI sample deposition. The results demonstrate that the sol-gel/crown ether hybrid materials effectively suppress the formation of sodiated oligonucleotides during MALDI analysis. The largest detectable molecular size for an oligonucleotide was a 100-mer, and the detection limit for an oligonucleotide 36-mer was ca. 20 fmol.     

The detection of intact double-stranded DNA by MALDI

DNA fragments have been analyzed by matrix-assisted laser desorption ionization (MALDI) and electrospray mass spectrometry. In many cases, only the single-stranded oligonucleotides have been detected. Recently, spectra of intact double-stranded DNA have been obtained in both electrospray and massive cluster impact ionization. We show here the first MALDI spectra of intact double-stranded DNA (EcoR1 adaptor 12/16) that is clearly not due to nonspecific dimer formation. 6-Aza-2-thiothymine was used as the matrix in the presence of ammonium citrate. Via the same procedure but with other matrices commonly employed for oligonucleotide analysis, the intact DNA duplex was not detected. No sign of the homodimer of either of the single strands is observed. Although the spectrum also shows peaks attributable to each of the single strands, these are demonstrated to arise from the DNA solution and not the sample preparation or desorption process.     

Formation and characterization of iron-oligonucleotide complexes with matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry

Iron-containing oligonucleotide negative ions can be generated by matrix-assisted laser desorption/ionization from a stainless steel target disk (by either defocusing the laser beam or by mixing iron salts such as FeCl3 with the matrix compound during the sample preparation). High resolution mass measurements reveal the presence of both Fe2+ (as M + Fe - 3H)- and Fe3+ (as M + Fe - 4H)- in the metal-oligonucleotide ions. The presence of Fe3+ is unexpected, and must involve replacement of protons from the nucleic bases or ribose groups as well as the phosphate groups of the oligonucleotides. Inspection of a range of small oligonucleotides and mononucleotides reveals that the presence of both Fe2+ and Fe3+ in the iron-biomolecule complexes is dependent on the number of acidic hydrogens that can be replaced in the oligonucleotide or nucleotide. Collisional dissociation of several metal-tetranucleotide ions revealed that the presence of the iron ion alters the fragmentation observed. The iron atom was observed to be present in all of the fragment ions, and, whenever possible, seemed to enhance the abundance of fragment ions containing both iron and a guanine nucleic base. These results suggest that iron may serve as a useful probe for characterizing phosphorylated biomolecules.     

Investigations of matrix-assisted laser desorption/ionization sample preparation by time-of-flight secondary ion mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) analyses are compared to gain insight into some of the details of sample preparation for MALDI analysis of synthetic polymers. ToF-SIMS imaging of MALDI samples shows segregation of the cationization agent from the matrix crystals. The amount of observed segregation can be controlled by the sample preparation technique. Electrospray sample deposition minimizes segregation. Comparing ToF-SIMS and MALDI mass spectra from the same samples confirms that ToF-SIMS is significantly more surface sensitive than MALDI. This comparison shows that segregation of the oligomers of a polymer sample can occur during MALDI sample preparation. Our data indicate that MALDI is not as sensitive to those species dominating the sample surface as to species better incorporated into the matrix crystals. Finally, we show that matrix-enhanced SIMS can be an effective tool to analyze synthetic polymers, although the sample preparation conditions may be different than those optimized for MALDI.     

Chemically induced hairpin formation in DNA monolayers

A naphthyridine dimer that binds specifically to G-G mismatches has been used to induce hairpin formation in oligonucleotides immobilized onto chemically modified gold surfaces. Surface plasmon resonance (SPR) imaging measurements of DNA microarrays were used to demonstrate that binding of the naphthyridine dimer to G-G mismatches within the stem portion of an immobilized 42-mer oligonucleotide could be used to induce hairpin formation that prevented hybridization of DNA complementary to the loop sequence. In addition, the selectivity of the naphthyridine dimer for G-G mismatches was verified through SPR imaging measurements of the hybridization adsorption of an 11-mer oligonucleotide to a four-component DNA array of zero- and single-base mismatch sequences.     

Application of DNA-binding polymers for preparation of DNA for analysis by matrix-assisted laser desorption/ionization mass spectrometry

The susceptibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the presence of salts in a sample, especially salts of alkali metals, requires careful and often tedious desalting procedures which complicate and slow the throughput of MS-based methods. A novel approach to sample preparation was developed based on the extraction of DNA out of solution onto a solid surface with an attached DNA-binding polymer, such as polyethyleneimine or polyvinylpyrrolidone. The observed binding is strong enough to sustain washing, and, as a result, desalting and concentration can be performed in a single fast step. After DNA has been immobilized on the surface and supernatant solution removed, subsequent addition of MALDI matrix releases material from the surface, which co-crystallizes with matrix. The mass spectrometric analysis is then performed directly from this support. Analysis of oligonucleotides and three-fold multiplexed SNP typing reactions performed by this method shows improved sensitivity and excellent resolution for various DNA fragments, together with high tolerance to various buffer components, such as alkali metals and surfactants. Simplicity and speed make it attractive for high-throughput sample preparation and analysis of oligonucleotide mixtures by MALDI-MS.     

In situ formation of chitosan-cyclodextrin nanospheres for drug delivery

Chitosan-cyclodextrin nanospheres were prepared by in situ formation through Michael addition between N-maleated chitosan (NMC) and per-6-thio-β-cyclodextrin sodium salt in an aqueous medium. This facile preparation method did not involve any organic solvent and surfactant. Through adjusting the preparation conditions, the nanospheres with a relatively narrow size distribution could be obtained. The obtained nanospheres were characterized by TEM and particle size analyzer. Doxorubicin hydrochloride (DOX·HCl), a water soluble anticancer drug, was loaded in the nanospheres with a high encapsulation efficiency. The in vitro drug release showed that the release of DOX·HCl from the nanospheres could be effectively sustained. The cytotoxicity evaluation showed the drug loaded nanospheres exhibited efficient inhibition on HeLa cells.     

Determining the molar mass of a plasma substitute succinylated gelatin by size exclusion chromatography-multi-angle laser light scattering,sedimentation equilibrium and conventional size exclusion chromatography

The clinical effectiveness of succinylated gelatin as a plasma substitute depends strongly on its molar mass, determined conventionally by size exclusion chromatography (SEC). This study evaluates different SEC calibration standards in comparison with two independent "absolute" methods for determining the weight average molar mass (M(w)) of a succinylated gelatin sample. SEC calibrated using succinylated gelatin fractions correlated well with size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) and sedimentation equilibrium whereas SEC calibrated with unmodified gelatin, sodium polystyrene sulfonates or pullulans overestimated M(w) by over 20%. Universal calibration was equivocal. The problems associated with the preparation of succinylated gelatin fractions suggest that an absolute method such as SEC-MALLS may be a more suitable choice for determining the M(w) in succinylated gelatins.     

Ultraviolet/matrix-assisted laser desorption/ionization mass spectrometric characterization of 2,5-dihydroxybenzoic acid-induced reductive hydrogenation of oligonucleotides on cytosine residues

The changes in the ion signals in the isotope cluster, mass resolution, signal-to-noise ratio and mass accuracy for matrix-assisted laser desorption/ionization (MALDI) of DNA oligonucleotides (dGGATC, dCAGCt, and dAACCGTT) and their fragment ions were evaluated, and these data were compared with those obtained using 3-hydroxypicolinic acid. Mass spectra obtained by using 2,5-dihydroxybenzoic acid (2,5-DHB) appear to have differences from the theoretical isotopic clusters, which arise by reductive hydrogenation producing a second peak at the M + 2 isotope of the native oligonucleotide. Based on the patterns of the isotopic envelope observed in the in-source decay fragments, we propose that cytosine is the site of reduction. We do not find evidence of reduction of oligonucleotides, viz. dTGGGGTT, that do not contain cytosine; however, 2'-deoxycytidine and 2'-deoxycytidine-5'-monophosphate undergo reductive hydrogenation. Several experiments were carried out in an effort to determine whether the reductive hydrogenation occurs during sample preparation or as a result of laser irradiation. The results of these experiments suggest that it occurs during sample preparation. The relative intensities of ion signals corresponding to the reduced base can be altered by using different matrix additives (aminonaphthalenes) or a different substrate (copper). Also, the oxidized form of 2,5-DHB is trapped by reaction with the side chain of cysteine in glutathione, providing evidence that the reaction occurs in solution as the matrix crystallizes.     

The effect of sodium dodecyl sulfate and anion‐exchange silica gel on matrix‐assisted laser desorption/ionization mass spectrometric analysis of proteins

Sodium dodecyl sulfate (SDS), an anionic surfactant, is widely used in peptide and protein sample preparation. When the sample is analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), this surfactant can often cause signal suppression. We have previously reported an on‐probe sample preparation method using a suspension of anion‐exchange silica gel and sinapinic acid (i.e., gel‐SA suspension) as a matrix, thereby greatly improving the MALDI signal detection of the protein solutions containing SDS. In this study, we found that a certain amount of SDS enhanced the MALDI signal intensity for protein samples. This effect was also observed when using sodium decyl sulfate and sodium tetradecyl sulfate instead of SDS. Furthermore, this on‐probe sample preparation method using both SDS and the gel‐SA suspension improved the detection limit of protein samples in the MALDI‐MS analysis by about ten‐fold as compared to that of protein samples without SDS and the gel‐SA suspension. This method can be applied not only to the MALDI‐MS analysis of samples containing SDS, but also to the examination of proteins at femtomole levels or insoluble proteins such as membrane proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Factors affecting atrazine concentration and quantitative determination in chlorinated water

Although the herbicide atrazine has been reported to not react measurably with free chlorine during drinking water treatment, this work demonstrates that at contact times consistent with drinking water distribution system residence times, a transformation of atrazine can be observed. Some transformation products detected through the use of high performance liquid chromatography–electrospray mass spectrometry are consistent with the formation of N-chloro atrazine. The effects of applied chlorine, pH, and reaction time on the transformation reaction were studied to help understand the practical implications of the transformation on the accurate determination of atrazine in drinking waters. The errors in the determination of atrazine are a function of the type of dechlorinating agent applied during sample preparation and the analytical instrumentation utilized. When a reductive dechlorinating agent, such as sodium sulfite or ascorbic acid is used, the quantification of the atrazine can be inaccurate, ranging from 2-fold at pH 7.5 to 30-fold at pH 6.0. The results suggest HPLC/UV and ammonium chloride quenching may be best for accurate quantification. Hence, the results also appear to have implications for both compliance monitoring and health effects studies that utilize gas chromatography analysis with sodium sulfite or ascorbic acid as the quenching agent.