Electrospray Ionization Time-of-flight Research Articles

The binding of guanine–guanine mismatched DNA to naphthyridine dimer immobilized sensor surfaces: kinetic aspects

Naphthyridine dimer composed of two naphthyridine chromophores and a linker connecting them strongly, and selectively, binds to the guanine–guanine mismatch in duplex DNA. The kinetics for the binding of the G–G mismatch to the naphthyridine dimer was investigated by surface plasmon resonance assay. The sensor surface was prepared by immobilizing naphthyridine dimer through a long poly(ethylene oxide) linker with the ligand density of 9.1 × 10 −12 fmol nm −2. The kinetic analyses revealed that the binding of the G–G mismatch was sequence dependent on the flanking base pairs, and the G–G mismatches flanking at least one G–C base pair bound to the surface via a two-step process with a 1:1 DNA–ligand stoichiometry. The first association rate constant for the binding of the G–G mismatch in the 5′-CGG-3′/3′-GGC-5′ sequence to the naphthyridine dimer-immobilized sensor surface was 3.2 × 10 3 M −1 s −1 and the first dissociation rate constant was 1.4 × 10 −2 s −1. The association and dissociation rate constants for the second step were insensitive to the flanking sequences, and were almost of the same order of magnitude as the first dissociation rate constant. This indicates that the second step had only a small energetic contribution to the binding. The association constant calculated from kinetic parameters was 2.7 × 10 5 M −1, which is significantly smaller than the apparent association constants obtained from experiments in solution. Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry on the complex produced from the G–G mismatch and naphthyridine dimer showed the formation of the 1:1 complex and a 1:2 DNA–ligand complex in solution. The latter complex became the dominant complex when a six-fold excess of naphthyridine dimer was added to DNA.

Exact mass measurement on an electrospray ionization time-of-flight mass spectrometer: error distribution and selective averaging.

An automated, accurate and reliable way of acquiring and processing flow injection data for exact mass measurement using a bench-top electrospray ionization time-of-flight (ESI-TOF) mass spectrometer is described. Using Visual Basic programs, individual scans were selected objectively with restrictions on ion counts per second for both the compound of interest and the mass reference peaks. The selected "good scans" were then subjected to two different data-processing schemes ("combine-then-center" and "center-then-average"), and the results were compared at various ion count limit settings. It was found that, in general, the average of mass values from individual scans is more accurate than the centroid mass value of the combined (same) scans. In order to acquire a large number of good scans in one injection (to increase the sampling size for statistically valid averaging), an on-line dilution chamber was added to slow down the typically rapid mass chromatographic peak decay in flow-injection analysis. This simple addition worked well in automation without the need for manual sample dilution. In addition, by dissolving the reference compound directly into the mobile phase, manual syringe filling can be eliminated. Twenty-seven samples were analyzed with the new acquisition and process routines in positive electrospray ionization mode. For the best method found, the percentage of samples with RMS error less than 5 ppm was 100% with repetitive injection data (6 injections per sample), and 95% with single injection data. Afterwards, 31 other test samples were run (with MW ranging from 310 to 3493 Da, 21 samples in ESI+ and 10 in ESI- mode) and processed with similar parameters and 100% of them were mass-calculated to RMS error less than 5 ppm also.

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique.

A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.

Cu(II)-catalyzed oxidation of beta-amyloid peptide targets His13 and His14 over His6: Detection of 2-Oxo-histidine by HPLC-MS/MS.

The interaction of beta-amyloid peptide (betaAP) with Cu(II) leads to the formation of reactive oxygen species, neurotoxicity, and the chemical modification of the peptide. To study product formation and the potential selectivity of oxidation, we have exposed specific betaAP congeners, betaAP1-16, betaAP1-28, and betaAP1-40, to ascorbate/Cu(II)-induced metal-catalyzed oxidation and electrospray ionization-time-of-flight (ESI-TOF) MS/MS analysis. Incubation of 30 microM betaAP with 15-150 microM Cu(II) and (physiologically relevant) 720 microM ascorbate in 20 mM phosphate buffer, pH 7.4, leads to significant oxidation of the peptides within remarkably short reaction times of as low as 6 min. Initial oxidation targets are His13 and His14, which are converted to 2-oxo-His, whereas the other two metal-binding residues, His6 and Tyr10, remain intact. Longer oxidation times then also target His6. Even in betaAP1-40 the oxidation of His13 and His14 precedes the oxidation of Met35. Especially, the insensitivity of Tyr10 is noteworthy and may be explained by electron withdrawal from the Tyr side chain through complexation of Cu(II). The insensitivity of His6 to initial oxidation may be rationalized by a proposed bridging of two Cu(II)-betaAP congeners, lowering the electron density on His6, comparable to similar results on a Cu(II)- and Zn(II)-bridging His61 residue of bovine Cu,Zn superoxide dismutase.

An alternative base-pairing of catechol-bearing nucleosides by borate formation.

A chelator-type beta-C-nucleoside having a catechol ligand as the nucleobase was found to form a stable 2:1 complex with trimethyl borate, which was characterized by 1H-NMR and electrospray ionization time-of-flight (ESI-TOF) mass spectroscopies. Both phosphoramidite and phosphotriester derivatives of this nucleoside were also prepared as synthetic precursors for DNA oligomer syntheses.

Identification of defensins in human lymphocyte nuclei.

The cell nucleus plays an essential role in all aspects of cell function and regulation. Most of the nuclear proteins/peptides are synthesized in the cytoplasm and transported into the nucleus through the nuclear pore complexes. The nuclear proteins/peptides conjugate with each other and interact in transcriptional activation/inactivation. Several of the high molecular mass transcription factors (> 30 kDa) have been identified and characterized. However, the information on the low molecular mass proteins/peptides of the nucleus is limited. We have investigated these low molecular mass proteins/peptides from the nucleus of human peripheral blood lymphocytes using reversed-phase high-performance liquid chromatography (RP-HPLC). The HPLC fractions were further analysed by matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, electrospray ionization time of flight (ESI-TOF) mass spectrometry and electrospray ionization fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry for mass determination. Using this combination of mass spectrometry techniques and microsequence analysis, we have shown that human lymphocyte nuclei contain defensins, a mixture of human neutrophil granule peptide 1, 2 and 3.

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.