Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide.

Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 µM up to 2.5 µM. The detection limit was 0.08 µM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 µM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.

Influence of Surface Chemistry on Host/Guest Interactions: A Model Study on Redox-Sensitive ß-Cyclodextrin/Ferrocene Complexes.

While host/guest interactions are widely used to control molecular assembly on surfaces, quantitative information on the effect of surface chemistry on their efficiency is lacking. To address this question, we combined electrochemical characterization with quartz crystal microbalance with dissipation monitoring to study host/guest interactions between surface-attached ferrocene (Fc) guests and soluble ß-cyclodextrin (ß-CD) hosts. We identified several parameters that influence the redox response, ß-CD complexation ability, and repellent properties of Fc monolayers, including the method of Fc grafting, the linker connecting Fc with the surface, and the diluting molecule used to tune Fc surface density. The study on monovalent ß-CD/Fc complexation was completed by the characterization of multivalent interactions between Fc monolayers and ß-CD-functionalized polymers, with new insights being obtained on the interplay between the surface chemistry, binding efficiency, and reversibility under electrochemical stimulus. These results should facilitate the design of well-defined functional interfaces and their implementation in stimuli-responsive materials and sensing devices.

LC-MS accurate quantification of a tryptic peptide co-eluted with an isobaric interference by using in-source collisional purification.

Interferences from isobaric and isomeric compounds represent a common problem in liquid chromatography coupled to mass spectrometry (LC-MS). In this paper, in-source purification and chromatographic separation were combined with the aim of identifying isobaric contamination and quantifying accurately a compound despite the presence of an isobaric co-eluted interference. This is achieved by totally fragmenting in-source the precursor ions of the isobaric interference providing then LC-pseudo-MS2 capability, which allows an accurate quantification without the need for optimizing the chromatographic conditions to separate the co-eluted interference. To illustrate this concept, mixtures of tryptic and non-tryptic peptides were used. The ratio of peak areas of the tryptic peptide and its isotopically labelled internal standard was used not only for quantification with an internal standard calibration curve but also to know (1) if an isobaric interference co-eluted with the tryptic peptide; and (2) what is the minimum cone voltage necessary to ensure the complete removal of isobaric interference. This strategy was applied to quantify the tryptic peptide of two standards with known concentrations and, intentionally contaminated with the isobaric interference. The confidence intervals of the concentrations calculated with the internal standard calibration curve were 8.0 ± 0.5 µM (prepared at 8.0 µM) and 15.7 ± 0.5 µM (prepared at 16.1 µM) that confirm the tryptic peptide can be correctly quantified by in-source purification without the need for improving the chromatographic separation from its isobaric interference.

Luminescent Ruthenium(II) Complexes Used for the Detection of 8-Oxoguanine in the Human Telomeric Sequence.

Detecting cancer at the early stage of the disease is crucial to keep the best chance for successful treatment. The recent development of genomic screening, a methodology that is addressed to asymptomatic patients presumably at risk of carcinogenesis, has stimulated the quest for new tools able to signal the level of risk. Carcinogenesis has been associated to chronic oxidative stress exceeding the antioxidant defenses and leading to critical genome alteration levels. The telomeric regions are presumably the most exposed to oxidative stress due to their high concentration of guanine (i.e., the easiest oxidizable nucleic base). Accumulation of 8-oxoguanine in telomeres, thus oxidative lesions, was reportedly associated with telomeric crisis and carcinogenesis. In this study, we report on the capacity of Ru(II) polyazaaromatic complexes to photoprobe 8-oxoguanine into the human telomeric sequence with the view of developing new tools for cancer risk screening.

Determination of the Rituximab Binding Site to the CD20 Epitope Using SPOT Synthesis and Surface Plasmon Resonance Analyses.

Antibodies not only play a major role in clinical diagnostics and biopharmaceutical analysis but also are a class of drugs that are regularly used to treat numerous diseases. The identification of antibody-epitope binding sites is then of great interest to many emerging medical and bioanalytical applications, particularly to design monoclonal antibodies (mAb) mimics taking advantage of amino acid residues involved in the binding. Among relevant antibodies, the monoclonal antibody rituximab has received significant attention as it is exploited to treat several cancers including non-Hodgkin's lymphoma and chronic lymphocytic leukemia, as well as some autoimmune disorders such as rheumatoid arthritis. The binding of rituximab to the targeted cells occurs via the recognition of the CD20 epitope. A crystallographic study has shown that the binding area, named paratope, is located at the surface of rituximab. Combining the SPOT method and the complementary surface plasmon resonance technique allowed us to detect an extended recognition domain buried in the pocket of the rituximab Fab formed by four ß-sheets. More generally, the present study offers a comprehensive approach to identify antibody-epitope binding sites.

Post-synthetic transamination at position N4 of cytosine in oligonucleotides assembled with routinely used phosphoramidites.

The commercially available and cheap nucleotide phosphoramidites are routinely used for the oligonucleotide (ODN) assembly. T, isobutyryl-dG (iBudG), benzoyl-dA (BzdA), acetyl-dC (AcdC) and benzoyl-dC (BzdC) derivatives are sufficient to produce orthogonally protected ODNs. Clean and efficient (ca. 30%-70% yield) post-synthetic amination of an ODN assembled with such phosphoramidites was selectively achieved at the N4 position of a singly introduced BzdC. Such a method represents a novel and cheap strategy for the user-friendly post-modification of oligonucleotides at the internal position.

Impact of Antigen Density on Recognition by Monoclonal Antibodies.

Understanding antigen-antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and nonmonotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition.

Quantification of intramolecular click chemistry modified synthetic peptide isomers in mixtures using tandem mass spectrometry and the survival yield technique.

Intramolecular click-chemistry is increasingly used to generate and control the architecture of complex macromolecules including peptides. Such compounds are, however, very challenging to analyze, in particular quantitatively and also to assess their purity. In this study, tandem mass spectrometry (MS/MS) experiments were carried out with an ion trap mass spectrometer using the Survival Yield (SY) technique to analyze several mixtures of protonated, alkali and alkaline earth metal complexes of two topological linear and cyclic peptide isomers. Univariate (using a single excitation voltage) and multivariate (using several excitation voltages) calibration models have been used. The sensitivity, linearity (R2), intermediate precision (sInt) and error of predicted values (RMSEP) of external calibrations curves have been compared leading to the conclusions that: 1) quantification using tandem mass spectrometry can be performed, with very good performances, for such peptides despite isomerism, 2) quantification is also possible despite the absence of diagnostic fragment ions (possibly independently of the amino-acid sequence), 3) best results are obtained with the largest alkali cation, Cs+, while protonation is highly discouraged, 4) uni/multivariate models show similar performances, but the univariate model may be more suitable for potential applications with direct infusion by electrospray ionization (ESI-MS/MS) and/or matrix-assisted laser desorption ionization (MALDI-MS/MS). Graphical abstract ᅟ.

Efficient Buchwald-Hartwig-Migita Cross-Coupling for DNA Thioglycoconjugation.

An efficient method for the thioglycoconjugation of iodinated oligonucleotides by Buchwald-Hartwig-Migita cross-coupling under mild conditions is reported. The method enables divergent synthesis of many different functionalized thioglycosylated ODNs in good yields, without affecting the integrity of the other A, C, and G nucleobases.

On the synthesis of cyclodextrin-peptide conjugates by the Huisgen reaction.

A,D-substituted cyclodextrin (CDX) substituted on their primary rim side are ideal scaffolds for the macromolecular assembly and formation of templated structures. Their substitution can be achieved through various reactions. However, the use of the well-known Huisgen reaction in this context is under-reported. We present here results of the synthesis of model conjugates formed between CDX and representative peptides by click chemistry. Notably, bis-conjugation of peptides onto a unique scaffold promotes α-helix formation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis of 5'-Aldehyde Oligonucleotide.

Synthesis of oligonucleotide ending with an aldehyde functional group at their 5'-end (5'-AON) is possible for both DNA (5'-AODN) and RNA (5'-AORN) series irrespectively of the nature of the last nucleobase. The 5'-alcohol of on-support ODN is mildly oxidized under Moffat conditions. Transient protection of the resulting aldehyde by N,N'-diphenylethylenediamine derivatives allows cleavage, deprotection, and RP-HPLC purification of the protected 5'-AON. Finally, 5'-AON is deprotected by usual acetic acid treatment. In the aggregates, 5'-AON can be now synthesized and purified as routinely as non-modified ODNs, following procedures similar to the well-known "DMT-On" strategy.

Parameters influencing the photo-induced electron transfer from tryptophan-containing peptides to a Ru(II) complex: a systematic study.

Ruthenium(II) polyazaaromatic complexes have gained interest in recent decades as biomolecular tools, especially in the development of new phototherapeutic agents. These light emissive Ru complexes based on π-deficient ligands were first designed to allow a photo-induced electron transfer (PET) with the guanine base in DNA since their (3)MLCT state is highly photo-oxidizing. Later the field of research was extended to proteins with the highlighting of a PET process with the tryptophan residue. This paper reports the kinetics of the luminescence quenching of [Ru(TAP)2phen](2+) by several selected peptide sequences containing at least one tryptophan residue. By using a peptide library we highlight the important parameters influencing the kinetics of the photo-electron transfer process, such as the net electrostatic charge and the number of tryptophan residues. The best peptide candidates were selected to study the formation of photo-products by MALDI-ToF mass spectrometry. A high photoreactivity of the [Ru(TAP)2phen](2+) complex was observed and multiple photoadducts were characterized, among them inter-peptidic adducts as well as intra-peptidic adducts.

Assessment of the Full Compatibility of Copper(I)-Catalyzed Alkyne-Azide Cycloaddition and Oxime Click Reactions for bis-Labelling of Oligonucleotides.

The conjugation of oligonucleotides with reporters is of great interest for improving their intrinsic properties or endowing new ones. In this context, we report herein a new procedure for the bis-labelling of oligonucleotides through oxime ligation (Click-O) and copper(I)-catalyzed alkyne-azide cycloaddition (Click-H). 5'-Azido and 3'-aldehyde precursors were incorporated into oligonucleotides, and subsequent coupling reactions through Click-O and Click-H (or vice versa) were successfully achieved. In particular, we exhaustively investigated the full compatibility of each required step for both tethering strategies. The results demonstrate that click Huisgen and click oxime reactions are fully compatible. However, whilst both approaches can deliver the targeted doubly conjugated oligonucleotide, the route involving click oxime ligation prior to click Huisgen is significantly more successful. Thus the reactions investigated here can be considered to be key elements of the chemical toolbox for the synthesis of highly sophisticated bioconjugates.

Synthetic access to the chemical diversity of DNA and RNA 5'-aldehyde lesions.

Hydrogen atom abstraction from the C5'-position of nucleotides in DNA results in direct strand scission by generating alkali-labile fragments from the oxidized nucleotide. The major damage consists in a terminus containing a 5'-aldehyde as part of an otherwise undamaged nucleotide. Moreover it is considered as a polymorphic DNA strand break lesion since it can be borne by any of the four nucleosides encountered in DNA. Here we propose an expeditious synthesis of oligonucleotides (ON) ending with this 5'-aldehyde group (5'-AODN). This straightforward and cheap strategy relies on Pfitzner­Moffatt oxidation performed on solid support followed by a transient protection of the resulting aldehyde function. This method is irrespective of the 5'-terminal nucleobase and most interestingly can be directly extended to RNA to produce the corresponding 5'-AORN. We also report preliminary results on recognition of 5'-AODN by base excision repair (BER) enzymes.

Preparation of azido containing oligonucleotides through diazo transfer reaction.

The use of diazo transfer reagent, imidazole-1-sulfonyl azide hydrochloride (ISAHC), in the presence of Cu(2+) cation enables clean and efficient conversion of aminated oligodeoxyribonucleotides (ODNs) into their azido counterparts under mild conditions. ODNs bearing amino tether either at the 3', 5', or any internal position could be modified in this way, thus demonstrating the versatility of this reaction. The method also benefits from the commercial availability or easy access by routine automated DNA synthesis of amino-containing oligodeoxyribonucleotide starting material. Easy access to such azido-modified ODNs is of great interest for conjugation in particular through copper catalyzed 1,3-dipolar cycloaddition (CuAAC reaction).

Phosphorylating reagent-free synthesis of 5'-phosphate oligonucleotides by controlled oxidative degradation of their 5'-end.

The 5'-phosphorylated oligonucleotides (5'-pONs) are currently synthesized using expensive and sensitive modified phosphoramidite reagents. In this work, a simple, cost-effective, efficient, and automatable method is presented, based on the controlled oxidation of the 5'-terminal alcohol followed by a ß-elimination reaction. The latter reaction leads to the removal of the terminal 5'-nucleoside and subsequent formation of the 5'-phosphate moiety. Thus, chemical phosphorylation of oligonucleotides (DNA or RNA) is achieved without using modified phosphoramidites.

Versatile introduction of azido moiety into oligonucleotides through diazo transfer reaction.

The use of a diazo transfer (DZT) reagent enables clean and efficient conversion of aminated oligodeoxyribonucleotides (ODNs) into their azido counterparts under mild conditions. ODNs bearing an amino tether at the 3', 5', or any internal position could be modified in this manner thus demonstrating the versatility of this reaction. Easy access to such azido-modified ODNs is of great interest for conjugation in particular through copper catalyzed 1,3-dipolar cycloaddition (CuAAC reaction).

Tandem mass spectrometric analysis of a mixture of isobars using the survival yield technique.

Collision induced dissociation tandem mass spectrometry experiments were performed to unequivocally separate compounds from an isobaric mixture of two products. The Survival Yield curve was obtained and is shown to consist in a linear combination of the curves corresponding to the two components separately. For such a mixture, a plateau appears on the diagram in lieu of the continuous decrease expected allowing for the structural study of the two components separately. The width of the plateau critically relates to the fragmentation parameters of the two molecular ions, which need to be sufficiently different structurally for the plateau to be observed. However, at constant fragmentation parameters, we have observed the width significantly increases at large m/z. This makes the separation more and more efficient as isobars have larger m/z and the technique complementary to those applicable at low m/z only. We have observed that the vertical position of the plateau relates linearly to the relative concentration of the two compounds that may be useful for quantification. Repeatability was estimated at 2% on a quadrupole ion trap. An advantage of using survival yield curves only, is that a priori knowledge of the respective fragmentation patterns of the two isobars becomes unnecessary. Consequently, similar performances are obtained if fragments are isobaric, which is also demonstrated in our study. The critical case of reverse peptides, at low m/z and similar fragmentation parameters, is also presented as a limitation of the method.

Trinaphthylamines as robust organic materials for two-photon-induced fluorescence.

The synthesis of a novel pi-conjugated trinaphthylamines series is described. These original push-pull octupolar systems exhibit large two-photon action cross section (sigma phi up to 510 GM) increased by a factor of 2-3 as compared to their triphenylamines analogues. This substantial improvement of the two-photon absorption properties is attributed to the stronger donor character of the trinaphthyl core.

Targeting DNA with "light-up" pyrimidine triple-helical forming oligonucleotides conjugated to stabilizing fluorophores (LU-TFOs).

The synthesis of triple-helix-forming oligonucleotides (TFOs) linked to a series of cyanine monomethines has been performed. Eight cyanines including one thiocyanine, four thiazole orange analogues, and three quinocyanines were attached to the 5'-end of 10-mer pyrimidine TFOs. The binding properties of these modified TFOs with their double-stranded DNA target were studied by absorption and steady-state fluorescence spectroscopy. The stability of the triplex structures depended on the cyanine structure and the linker size used to connect both entities. The most efficient cyanines able to stabilize the triplex structures, when attached at the 5'-end of the TFO, have been incorporated at both ends and provided triplex structures with increased stability. Fluorescence studies have shown that for the TFOs involving one cyanine, an important intensity increase (up to 37-fold) in the fluorescent signal was observed upon their hybridization with the double-stranded target, proving hybridization. The conjugates involving thiazole orange attached by the benzothiazole ring provided the most balanced properties in terms of triplex stabilization, fluorescence intensity and fluorescence enhancement upon hybridization with the double-stranded target. In order to test the influence of different parameters such as the TFO sequence and length, thiazole orange was used to label 17-mer TFOs. Hybridizations of these TFOs with different duplexes, designed to study the influence of mismatches at both internal and terminal positions on the triplex structures, confirmed the possibility of triplex formation without loss of specificity together with a strong fluorescence enhancement (up to 13-fold).