Electrospray Ionization Time-of-flight Research Articles

Cu(II) and Zn(II) crystal complexes based on pyrazolone: Synthesis and application as antibacterial agents

4-Benzoyl-3-methyl-1-phenyl-5-pyrazolone (HPMBP) was synthesized via the Friedel–Crafts reaction and characterized through 1H NMR spectroscopy, Infrared spectroscopy and Electrospray ionization time-of-flight(ESI-TOF). The Cu(II) and Zn(II) coordination complexes, namely, [Cu(HPMBP)(2,2′-bpy)(Cl)]·H2O(1) and [Zn(HPMBP)(2,2′-bpy)(Cl)]·H2O(2), were synthesized in CH3OH(HPMBP:2,2′-bipyridine:M = 1:1:1, M = Cu, Zn). Single crystal X-ray diffraction(SCXRD) studies show that Cu(II) and Zn(II) exhibit a distorted square pyramidal configuration through two oxygen atoms of HPMBP, two cis-nitrogen atoms of 2,2′-bipyridine and monodentate chloride ion. Moreover, the Hirshfeld surface analysis of the in complexes was performed using crystallographic calculations. Furthermore, the antibacterial activities of 1 and 2 are more potent than HPMBP, in particular, the antimicrobial properties of 2 are better than those of 1. Scanning electron microscopy (SEM) indicated that 1 had a regular strip shape, while 2 had an irregular circular shape. Our work provides a theoretical basis for studying the antibacterial activity of transition metal ion complexes.

Contrasting Bonding Extremes in Two [Ge6]n- Complexes: A Wadian Polyhedron (n = 2) versus a Hydrocarbon-like 2c-2e Polygermanide (n = 12).

[Nb(η6-C6H3Me3)2] reacts with ethylenediamine (en) solutions of K4Ge9 in the presence of 18-crown-6 to give [(η6-C6H3Me3)NbHGe6]2- (1) and [(η6-C6H3Me3)NbGe6Nb(η6-C6H3Me3)]2- (2) as their corresponding [K(18-crown-6)]+ salts. The crystalline solids are dark brown, air-sensitive, and sparingly soluble or insoluble in most solvents. The [K(18-crown-6)]+ salts of cluster ions 1 and 2 have been characterized by energy-dispersive X-ray (EDX) analysis, NMR studies, single-crystal X-ray diffraction, and electrospray ionization time-of-flight (ESI-TOF) mass spectrometry studies. Cluster ions 1 and 2 have markedly different [Ge6] moieties: an electron-deficient carborane-like subunit in 1 and a two-center, two-electron cyclohexane-like subunit in 2.

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

One-Pot Synthesis of Thermoresponsive Amyloidogenic Peptide-Polymer Conjugates via Thio-Bromo "Click" Reaction of RAFT Polymers.

A synthetic strategy to efficiently prepare main-chain peptide-polymer conjugates probing their aggregation in solution is described. An in situ tandem reaction based on aminolysis/thio-bromo "click" reaction is performed to tether an amyloidogenic peptide fragment amyloid-β17-20 (Leu-Val-Phe-Phe (LVFF)) to the ω-chain end of poly(diethylene glycol methyl ether acrylate) (PDEGA), prepared via reversible addition fragmentation chain transfer polymerization. Structural confirmation of the constructed conjugates PDEGA-LVFF (Mn,SEC = 5600, Ð = 1.21), (Mn,SEC = 7600, Ð = 1.16), and (Mn,SEC = 8900, Ð = 1.15) is successfully made by combined studies of 1 H NMR, size-exclusion chromatography, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. The effect of the peptidic constituent on the thermoresponsive behavior of the polymer is examined by UV-vis spectroscopy, and the self-assembly behavior of the amphiphilic conjugate is further exploited, exhibiting micellar morphology in aqueous solution.

Analysis of oxovanadium(IV) complexes using electrospray mass spectrometry: Molecular ion formed by loss of an electron, and its product ions trapping oxygen.

In addition to their biological properties, oxovanadium complexes have been widely applied as catalysts because of their excellent catalytic-oxidation capability. Recently, monometallic oxovanadium(IV) complexes have been used as catalysts in the electrophilic trifluoromethylation of silyl ketene imines. The study of catalysts can contribute to an understanding of the reaction mechanism. Six monometallic oxovanadium(IV) complexes were analyzed by electrospray ionization time-of flight mass spectrometry (ESI-TOFMS), and collision-induced dissociation mass spectrometry (CID-MS) experiments were conducted for selected cations [M]+ of oxovanadium(IV) complexes as well as a deuterium-labeled complex. Different collision gases were used to understand the source of the O2 and H2 O engaged in the gas-phase ion-molecule reaction. The oxovanadium(IV) complexes formed [M]+ ions by loss of an electron, with [M+14]+ ions being formed from [M]+ by loss of H2 O and addition of O2 . The fragmentation pathways of the [M]+ cations were further studied by ESI-MS/MS, and several ions produced by gas-phase ion-molecule reactions were detected and characterized, including vanadium-oxo, -peroxo and derivatives. Several unexpected ions were detected, including [M]+ , [M+14]+ and ions produced from gas-phase ion-molecule reactions. The study has contributed to the understanding of the structure and character of oxovanadium(IV) complexes, and it could facilitate the design of new oxovanadium catalysts and an understanding of their reaction mechanism.

Critical Conditions for Liquid Chromatography of Statistical Copolymers: Functionality Type and Composition Distribution Characterization by UP-LCCC/ESI-MS.

Statistical ethylene oxide (EO) and propylene oxide (PO) copolymers of different monomer compositions and different average molar masses additionally containing two kinds of end groups (FTD) were investigated by ultra high pressure liquid chromatography under critical conditions (UP-LCCC) combined with electrospray ionization time-of flight mass spectrometry (ESI-TOF-MS). Theoretical predictions of the existence of a critical adsorption point (CPA) for statistical copolymers with a given chemical and sequence distribution1 could be studied and confirmed. A fundamentally new approach to determine these critical conditions in a copolymer, alongside the inevitable chemical composition distribution (CCD), with mass spectrometric detection, is described. The shift of the critical eluent composition with the monomer composition of the polymers was determined. Due to the broad molar mass distribution (MMD) and the presumed existence of different end group functionalities as well as monomer sequence distribution (MSD), gradient separation only by CCD was not possible. Therefore, isocratic separation conditions at the CPA of definite CCD fractions were developed. Although the various present distributions partly superimposed the separation process, the goal of separation by end group functionality was still achieved on the basis of the additional dimension of ESI-TOF-MS. The existence of HO-H besides the desired allylO-H end group functionalities was confirmed and their amount estimated. Furthermore, indications for a MSD were found by UPLC/MS/MS measurements. This approach offers for the first time the possibility to obtain a fingerprint of a broad distributed statistical copolymer including MMD, FTD, CCD, and MSD.

Trifasciatosides A-J, Steroidal Saponins from Sansevieria trifasciata.

Four previously unreported steroidal saponins, trifasciatosides A-D (1-4), three pairs of previously undescribed steroidal saponins, trifasciatosides E-J (5a, b-7a, b) including acetylated ones, together with twelve known compounds were isolated from the n-butanol soluble fraction of the methanol extract of Sansevieria trifasciata. Their structures were elucidated on the basis of detailed spectroscopic analysis, including (1)H-NMR, (13)C-NMR, (1)H-(1)H correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity (HMBC), total correlated spectroscopy (TOCSY), nuclear Overhauser enhancement and exchange spectroscopy (NOESY), electrospray ionization-time of flight (ESI-TOF)-MS and chemical methods. Compounds 2, 4, and 7a, b exhibited moderate antiproliferative activity against HeLa cells.

Photoinduced Dimerization Reaction Coupled with Oxygenation of a Platinum(II)–Hydrazone Complex

Photoreactivities of Ni(II)- and Pt(II)-hydrazone complexes, [NiCl(L)] (Ni1) and [PtCl(L)] (Pt1), respectively [HL = 2-(diphenylphosphino)benzaldehyde-2-pyridylhydrazone], were investigated in detail via UV-vis absorption, (1)H nuclear magnetic resonance (NMR) spectroscopy, and electrospray ionization time-of-flight (ESI-TOF) mass spectrometry; the two photoproducts obtained from the photoreaction of Pt1 were also successfully identified via X-ray analysis. The absorption bands of the Ni1 and Pt1 complexes were very similar, centered around 530 nm, and were assigned as an intraligand charge transfer transition of the hydrazone moiety. The absorption spectrum of Pt1 in a CH3CN solution changed drastically upon photoirradiation (λ = 530 nm), whereas no change was observed for Ni1. (1)H NMR and ESI-TOF mass spectra under various conditions suggested that the photoexcited Pt1* reacts with dissolved dioxygen to form a reactive intermediate, and the ensuing dark reactions afforded two different products without any decomposition. In contrast to the simple photo-oxidation of HL to form a phosphine oxide HL(P═O), the X-ray crystallographic analyses of the photoproducts clearly indicate the formation of a mononuclear Pt complex with the oxygenated hydrazone ligand (Pt1O) and a dinuclear Pt complex with the oxygenated and dimerized hydrazone ligand (Pt2). The photosensitized reaction in the presence of an (1)O2-generating photosensitizer, methylene blue (MB), also produced Pt1O and Pt2, indicating that the reaction between (1)O2 and ground-state Pt1 is the important step. In a highly viscous dimethyl sulfoxide solution, Pt1 was slowly, but quantitatively, converted to the mononuclear form, Pt1O, without the formation of the dinuclear product, Pt2, upon photoirradiation (and in the reaction photosensitized by MB), suggesting that this photoreaction of Pt1 involves at least one diffusion-controlled reaction. On the other hand, the same complexes Pt1O and Pt2 were also produced in the degassed solution, probably because of the reaction of the photoexcited Pt1* with the biradical character and H2O.

Repair of DNA Alkylation Damage by the Escherichia coli Adaptive Response Protein AlkB as Studied by ESI‐TOF Mass Spectrometry

DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II)-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. In an effort to understand the range of acceptable substrates for AlkB, the enzyme was incubated with chemically synthesized oligonucleotides containing alkyl lesions, and the reaction products were analyzed by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. Consistent with the literature, but studied comparatively here for the first time, it was found that 1-methyladenine, 1,N 6-ethenoadenine, 3-methylcytosine, and 3-ethylcytosine were completely transformed by AlkB, while 1-methylguanine and 3-methylthymine were partially repaired. The repair intermediates (epoxide and possibly glycol) of 3,N 4-ethenocytosine are reported for the first time. It is also demonstrated that O 6-methylguanine and 5-methylcytosine are refractory to AlkB, lending support to the hypothesis that AlkB repairs only alkyl lesions attached to the nitrogen atoms of the nucleobase. ESI-TOF mass spectrometry is shown to be a sensitive and efficient tool for probing the comparative substrate specificities of DNA repair proteins in vitro.

Isolation of Carignan and Merlot red wine oligosaccharides and their characterization by ESI-MS

The oligosaccharides of Carignan and Merlot wines have been characterized for the first time. In a first step, this fraction was prepared after discoloration of the wines and was collected by elution on an HPSEC system. In a second step, the glycosyl composition and linkages of wine oligosaccharides were determined by several methods. High resolution MS spectra of the Carignan and Merlot oligosaccharide fractions were obtained on an AccuTOF mass spectrometer equipped with an electrospray ionization (ESI) source and a time-of-flight (TOF) mass analyser. Oligosaccharides were present at a concentration of 330 and 250 mg/L in Carignan and Merlot red wines, respectively. Glycosyl residue composition analysis showed the presence of mannose, arabinose, galactose, rhamnose, fucose, xylose, glucose, galacturonic acid, and glucuronic acid. We show that oligosaccharides are present in significant amounts in wines, that they result from the degradation of cell wall polysaccharides and that they have an extreme diversity, about 30 peaks in ESI–TOF spectra corresponding each to at least one oligosaccharidic structure. The ESI–TOF spectra in negative mode of the Carignan and Merlot oligosaccharides showed oligosaccharidic structures corresponding to oligogalacturonic acids, partially esterified by methyl group (trigalacturonic acid detected at m/ z 545, m/ z 559 and m/ z 573) or to the repetition of the basic unit [→4)-α- d-GalA p-(1→2)-α- l-Rha p-(1→] two ( m/ z 661), or three ( m/ z 983) times. These units can be substituted either by a hexose, or by a pentose, or by both, but also by deoxyhexose or uronic acid. The identification of [4-OMe-GlcA-[Xyl] 2-Xylitol] and [4-OMe-GlcA-[Xyl] 3-Xylitol] by MS n fragmentation performed on a mass spectrometer equipped with an ESI source and an ion trap mass analyser makes it possible to explain the presence of xylose in wines.

First Systematic MALDI/ESI Mass Spectrometry Comparison to Characterize Polystyrene Synthesized by Different Controlled Radical Polymerizations

Mass spectrometry techniques, specifically matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization time-of-flight (ESI-TOF) are of considerable interest for characterizing very attractive polymers resulting from controlled radical polymerizations (CRP). This work proposes a systematic and experimental comparison of both ionization methods to analyze polystyrene prepared by five different CRP: nitroxide-mediated polymerization (mediated by N-tert-butyl-N-[1-diethylphosphono-(2,2-dimethylpropyl)]nitroxide, known as SG1), atom transfer radical polymerization (Cl- and Br-mediated), reversible addition-fragmentation chain transfer polymerization, and reverse iodine transfer polymerization. All polystyrene chain end-groups obtained in each CRP-polymerization, and detected by both mass spectrometry techniques, are described and discussed. Elemental analyses of all CRP-polymers were also carried out to complete these results. It was found that the number of chain end-gro...

Mass measurement accuracy comparisons between a double‐focusing magnetic sector and a time‐of‐flight mass analyzer

We report a direct comparison of the mass measurement accuracies (MMAs) obtained on different mass spectrometry instrument types; a magnetic sector as the 'gold standard' and an electrospray ionization time-of-flight (ESI-TOF) instrument. Sixty samples, obtained from the Department of Chemistry at North Carolina State University, were analyzed on each instrument. Data are presented and compared between the different instruments. The average absolute MMAs achieved for the magnetic sector and Agilent ESI-TOF mass spectrometers were 3.0 and 1.1 ppm, respectively.

Development of a comprehensive multidimensional liquid chromatography system with tandem mass spectrometry detection for detailed characterization of recombinant proteins

A multidimensional comprehensive liquid-phase separation system (2DLC) coupled on-line to an electrospray-ionization time-of-flight (ESI-TOF) mass spectrometer (MS) was used to resolve structural alterations and/or post-translational modifications for detailed protein characterization. The system described in this work consists of cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. A unique spiked gradient was employed in the first dimension to enhance recovery of peptides. This combination of separation followed by MS detection offered the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. During the course of this study it was determined that altered or modified peptides were shown to be better resolved than during a one-dimensional separation. The 2DLC/ESI methodology allowed for a comprehensive evaluation of post-translational modifications and chemical reactions of recombinant proteins, providing a meaningful evaluation of product quality that was not possible with other current analytical approaches. In addition, the system can be used to provide sequence coverage of complex proteins.

Steady-State Asymmetric Nanospray Dual Ion Source for Accurate Mass Determination within a Chromatographic Separation

Here we report the design, implementation, and initial use of an asymmetric steady-state continuous dual-nanospray ion source. This new source design consists of two independently controlled and continuously operating nanospray interfaces with funnel shaped counter electrodes. A steady-state ion mixing region combines the ions from the two sources into a single ion beam in the intermediate region after ion extraction from the nanospray sources but before the bulk of the pressure gradient of the vacuum interface. With this design we have achieved robust mixing of ions with no loss of duty cycle and remarkable ionization characteristics that appear entirely noncompetitive and potentially beneficial. This allows continuous introduction of internal mass calibration ions during a liquid chromatography-mass spectrometric analysis. This in turn allows for recalibration of individual spectra yielding sub part per million mass accuracy throughout the run. The steady-state approach presented here has several advantages over previous approaches. Since neither the voltage nor positions of the sprayers are changed, the nanospray has greater spray stability. The ions produced by the analyte sprayer are continuously sampled, as opposed to time-sharing which necessitates that the analyte ion stream be interrupted for some part of the duty cycle. There are no moving parts, no rapid changes to high voltages requiring additional control electronics, and no need for completely separate vacuum interfaces and the associated complexity. The sprayers are independently controlled and do not exhibit competition or mutual ionization suppression. This novel source has been implemented with a Bruker Apex II 9.4 T FTICR with a modified Apollo electrospray ion source as part of a nanoflow liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry analysis platform. Because of the low cost of implementation, the new source could potentially be applied to other forms of mass spectrometry, such as electrospray ionization-time-of-flight (ESI-TOF), which can benefit from internal mass calibration.

Automated Sub-ppm Mass Accuracy on an ESI-TOF for Use with Drug Discovery Compound Libraries

An automated, routine method to obtain sub-ppm accurate mass data on a benchtop electrospray ionization time-of-flight (ESI-TOF) mass spectrometer is described. Standards in the mass range 114 to 734 Da were analyzed over a 5-day period to demonstrate intra- and interday precision and mean mass accuracy less than 1 ppm. One hundred drug discovery pharmaceutical compounds were used to demonstrate an absolute average mass accuracy of 0.47 +/- 0.31 ppm. This is in contrast to previous reports of accurate mass analysis using time-of-flight mass spectrometry (TOFMS) technology that operates within 3 to 5 ppm. The same 100 samples were also analyzed using Fourier transform mass spectrometry (FTMS) technology and yielded comparable results to the TOFMS analysis.

Triethylene Glycol Ether End-grafted Carbosilane Dendrimer: A Potential Ionophore for Potassium Ion Recognition

The performance of a newly synthesized carbosilane dendrimer bearing four triethylene glycol ether (TEG) units, Si(CH2CH2CH2Si(Me)2CH2CH2CH2(OCH2CH2)3Me)4 (1), as ionophores in ion-selective electrodes has been investigated. Optimization of the plasticized polyvinyl chloride membrane composition has produced electrodes that exhibit a Nernstian response for potassium ions. The best general characteristics exhibited by the electrodes were found when the membrane composition ratio of DPE:1:NaTPB:PVC 60:3:2:35 (wt%) was used. The response of the electrode was linear with a Nernstian slope of 58.3 mV/decade over the K+ ion concentration range of 1.9x10(-7) to 1.0x10(-1) M with a detection limit of 3.1x10(-7) M. The response time to achieve a 95% steady potential for the K+ concentration ranging from 1.0x10(-1) to 1.0x10(-8) M was less than 10 s, and it was found that the electrode is suitable for use within a pH range of 5.5-8.5. The selectivity coefficients (log KPotK+,Mn+)), which were determined by the fixed interference method, showed good selectivity for K+ against most of the interfering cations. The influence of this selective ion-binding behavior using electrospray ionization time-of-flight (ESI-TOF) mass spectrometric studies is discussed.

Isolation and purification of Pseudostellarin B (cyclic peptide) from Pseudostellaria heterophylla (Miq.) Pax by high-speed counter-current chromatography

Pseudostellarin B (cyclic peptide) was isolated and purified from the herbs of Pseudostellaria heterophylla (Miq.) Pax for the first time by high-speed counter-current chromatography (HSCCC) using a two-phase solvent system consisting of n-butanol–ethyl acetate–water (0.6:4.4:5, v/v). The technique can isolate mg levels of the target compound per run with purity better than 96%. The chemical structure of the compound has been positively confirmed by electrospray ionization time of flight (TOF) MS, 1H NMR, 13C NMR and 1H– 13C COSY analyses.

Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability

An enhanced analytical RP-HPLC/MS method was developed for monitoring the stability and production of intact and fragmented monoclonal antibodies (MAbs). The use of high column temperatures (70–80 °C), organic solvents with high eluotropic strength coefficients (isopropyl and n-propyl alcohols), and Zorbax StableBond columns, were critical for good recovery and resolution of immunoglobulin G1 (IgG1) and IgG2 monoclonal antibodies. Using this method, cleavage products of a degraded IgG1 antibody were clearly separated and identified by in-line electrospray ionization time-of-flight (ESI-TOF) mass spectrometry generating exact masses and unique terminal ladder sequences. The glycosylation profile, including mapping of the terminal galactose and fucose heterogeneity of the N-linked sugars, was determined by mass spectrometry of intact MAbs. In addition, we discovered that several IgG2 MAbs exhibited greater structural heterogeneity compared to IgG1s. Mass spectral characterization data and reduction data suggested that the heterogeneity is disulfide related. This reversed-phase LC/MS method represents a key advancement in monitoring intact MAb production and stability.

Prospective Evaluation of Graft-Versus-Host Disease-Specific Proteomics Pattern in Patients after Allogeneic Stem Cell Transplantation.

We have recently described and published a proteomic pattern specific for the early diagnosis of acute graft versus host disease (aGvHD), based on the application of capillary electrophoresis (CE) and mass spectrometry (MS). Here we report the application of proteome based GvHD-patterns to prospectively collected samples from 86 patients (45 AML, 14 ALL, 8 MM, 5 CLL, 5 SAA, 3 MDS, 3CML, 3 NHL). Fifty three patients were transplanted from matched unrelated donors (MUD), 29 received stem cells from matched related donors (MRD), 3 from haplo-identical donors and 1 was transplanted from a syngeneic sibling. GvHD prophylaxis was methotrexat or mycophenolate and cyclosporin A in the majority of the patients. The follow up is currently between 60 and 549 days after allogeneic HSCT. Urine samples were collected on ice prior conditioning, once a week and monthly after day +60. To avoid degradation of the proteins/peptides the samples were frozen as soon as possible. After thawing and removal of confounding substances, like salts, lipids and of all molecules larger than 30 kDa, the samples were loaded onto the CE, separated according to their charge and, after ionization, directly analyzed in an electrospray ionization time of flight (ESI-TOF) -MS. Between 500 and 2500 peptides and proteins were detected in individual samples. All data generated are stored in a Microsoft MS database. Reproducibility of these analyses is greater 98%. The polypeptide patterns specific for the early detection of acute GvHD were applied to the data generated prospectively and the outcome of these analyses was compared to the clinical diagnosis of aGvHD, sepsis and CMV-reactivation. Forty patients were diagnosed with aGvHD in the current evaluation period, 18 had aGvHD ≥ II. From 461 samples, 68 gave a score with the aGvHD pattern, 15 were false positive, whereas 5 scored false negative. The sensitivity of the aGvHD pattern is more than 90%, the specificity is about 96%. Thus the application of the aGvHD pattern for early recognition of acute GvHD is very useful for predicting the development of aGvHD. In addition, we are currently evaluating samples of patients with chronic GvHD (cGvHD). Seven patients in the prospective cohort have developed cGvHD so far. First results show that in the majority of the patients the cGVHD markers are different from those forming the aGvHD pattern. The polypeptide patterns for aGvHD and cGvHD will be discussed together with those of other complications, like CMV-reactivation or sepsis.Taken together our results demonstrate that the proteome analysis of body fluids collected from patients after HSCT maybe extremely useful for diagnosis of complications.

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

The kinetics for the binding of the G–G mismatch to the naphthyridine dimer was investigated by surface plasmon resonance assay. 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.