Cyclotribenzylene alkynylgold(I) phosphine complexes: synthesis, chirality, and exchange of phosphine.

Two different alkynyl-substituted C3-symmetric cyclotribenzylenes (CTB) were synthesized in racemic and enantiomerically pure forms, and six gold(I) phosphine complexes differing by the nature of the CTB and the phosphine were prepared and characterized, in particular by NMR spectroscopy, DOSY, electronic circular dichroism (ECD), and electrospray ionization mass spectrometry (ESI-MS). Their ECD patterns depended on the substitution of the starting CTBs and were shifted bathochromically by comparison with the latter. ESI-MS in the presence of HCO2H allowed us to detect the complexes as proton adducts. The intensities of the signals were stronger when the phosphine was more electron-rich. This technique was also used to investigate the exchange of phosphine betweeen pairs of CTB complexes. The scrambling reaction was demonstrated by the higher intensity of the signals of the complexes subjected to the exchange of a single phosphine ligand by comparison with the intensity of the signals of the starting complexes.

Hyphenation of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with separation methods: The art of compromises and the possible - A review.

This review provides an overview of the online hyphenation of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) with separation methods to date. The online coupling between separation techniques (gas and liquid chromatography, capillary electrophoresis) and FT-ICR MS essentially raises questions of compromise and is not look as straightforward as hyphenation with other analyzers (QTOF-MS for instance). FT-ICR MS requires time to reach its highest resolving power and accuracy in mass measurement capabilities whereas chromatographic and electrophoretic peaks are transient. In many applications, the strengths and the weaknesses of each technique are balanced by their hyphenation. Untargeted "Omics" (e.g. proteomics, metabolomics, petroleomics, …) is one of the main areas of application for FT-ICR MS hyphenated to online separation techniques because of the complexity of the sample. FT-ICR MS achieves the required high mass measurement accuracy to determine accurate molecular formulae and resolution for isobar distinction. Meanwhile separation techniques highlight isomers and reduce the ion suppression effects extending the dynamic range. Even if the implementation of FT-ICR MS hyphenated with online separation methods is a little trickier (the art of compromise), this review shows that it provides unparalleled results to the scientific community (the art of the possible), along with raising the issue of its future in the field with the relentless technological progress.

Nucleos'ID: A New Search Engine Enabling the Untargeted Identification of RNA Post-transcriptional Modifications from Tandem Mass Spectrometry Analyses of Nucleosides.

As RNA post-transcriptional modifications are of growing interest, several methods were developed for their characterization. One of them established for their identification, at the nucleosidic level, is the hyphenation of separation methods, such as liquid chromatography or capillary electrophoresis, to tandem mass spectrometry. However, to our knowledge, no software is yet available for the untargeted identification of RNA post-transcriptional modifications from MS/MS data-dependent acquisitions. Thus, very long and tedious manual data interpretations are required. To meet the need of easier and faster data interpretation, a new user-friendly search engine, called Nucleos'ID, was developed for CE-MS/MS and LC-MS/MS users. Performances of this new software were evaluated on CE-MS/MS data from nucleoside analyses of already well-described Saccharomyces cerevisiae transfer RNA and Bos taurus total tRNA extract. All samples showed great true positive, true negative, and false discovery rates considering the database size containing all modified and unmodified nucleosides referenced in the literature. The true positive and true negative rates obtained were above 0.94, while the false discovery rates were between 0.09 and 0.17. To increase the level of sample complexity, untargeted identification of several RNA modifications from Pseudomonas aeruginosa 70S ribosome was achieved by the Nucleos'ID search following CE-MS/MS analysis.

Optimization of nucleotides dephosphorylation for RNA structural characterization by tandem mass spectrometry hyphenated with separation methods.

As part of RNA characterization, the identification of post-transcriptional modifications can be performed using hyphenation of separation methods with mass spectrometry. To identify RNA modifications with those methods, a first total digestion followed by a dephosphorylation step are usually required to reduce RNA to nucleosides. Even though effective digestion and dephosphorylation are essential to avoid further complications in analysis and data interpretation, to our knowledge, no standard protocol is yet referenced in the literature. Therefore, the aim of this work is to optimize the dephosphorylation step using a total extract of transfer RNA (tRNA)1 from B. taurus as a model and to determine and fix two protocols, leading to complete dephosphorylation, based on time and bacterial alkaline phosphatase (BAP)2 consumptions. Capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) was used to estimate the dephosphorylation efficiency of both protocols on many canonical and modified nucleotides. For a timesaving protocol, we established that full dephosphorylation was obtained after a 4-hour incubation at 37 °C with 7.5 U of BAP for 1 µg of tRNA. And for a BAP-saving protocol, we established that full dephosphorylation was obtained 3.0 U of BAP after an overnight incubation at 37 °C. Both protocols are suitable for quantitative analyses as no loss of analytes is expected. Moreover, they can be widely used for all other RNA classes, including messenger RNA or ribosomal RNA.

Protocol for Etching Bare-Fused Silica Capillaries for Sheathless Capillary Electrophoresis-Mass Spectrometry Coupling.

Homemade capillaries are a very common practice for the users of capillary electrophoresis (CE), notably in CE-UV. With the advent of the capillary electrophoresis-mass spectrometry coupling since the end of the 1980s, several interfaces have been developed. Among those interfaces, the porous tip sprayer allows great sensitivity at nano flow rates and has been used in numerous applications over the past few years. However, the homemade implementation of a suitable capillary for the porous tip sprayer is more challenging. The porous tip is created by etching the bare-fused silica capillary with hydrofluoric acid. Here we describe the complete process of etching bare-fused silica capillaries, from length cutting to quality control of the newly etched capillary.

Alkynylgold(I) C3 -Chiral Concave Complexes: Aggregation and Luminescence.

Chiral gold(I) acetylide trinuclear complexes 1-3 based on the cyclotribenzylene platform and terminal PR3 ligands (R=Ph, Et, and Cy, respectively), were characterized and their light emission studied. They exhibited long-lived blue phosphorescence in CHCl3 and a weak fluorescence in the UV. In MeOH/CHCl3 mixtures of >1:1 volume ratio, 1 and 2 exhibited a new emission band at ca. 540 nm that developed at the expense of the UV emission. DLS studies demonstrated the presence of molecular aggregates of Ø 30-80 nm. The green emission observed in MeOH-rich solvent mixtures was therefore induced by aggregation, and could originate from Au⋅⋅⋅Au interactions. The AIE spectrum of 3 was observed only in solutions containing 99 % of MeOH, and correlated with its solid state emission. The AIE profiles of the enantiomers of 1 differed from that of rac-1, suggesting that the latter is a true racemate.

Analysis of Monoclonal Antibody Glycopeptides by Capillary Electrophoresis-Mass Spectrometry Coupling (CE-MS).

Glycosylation is a crucial posttranslational modification (PTM) that might affect the safety and efficacy of monoclonal antibodies (mAbs). Capillary electrophoresis-mass spectrometry (CE-MS) enables the characterization of the primary structure of mAbs. A bottom-up proteomic workflow is designed to provide detailed information about the glycosylation. In this chapter, we describe the validated experimental protocol applied for the characterization and relative quantification of mAbs N-glycosylation at the glycopeptide level.

Characterisation of hydrocarbon degradation, biosurfactant production, and biofilm formation in Serratia sp. Tan611: a new strain isolated from industrially contaminated environment in Algeria.

A novel bacterial strain was isolated from industrially contaminated waste water. In the presence of crude oil, this strain was shown to reduce the rate of total petroleum hydrocarbons (TPH) up to 97.10% in 24 h. This bacterium was subsequently identified by 16S rRNA gene sequence analysis and affiliated to the Serratia genus by the RDP classifier. Its genome was sequenced and annotated, and genes coding for catechol 1,2 dioxygenase and naphthalene 1,2-dioxygenase system involved in aromatic hydrocarbon catabolism, and LadA-type monooxygenases involved in alkane degradation, were identified. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of crude oil after biological treatment showed that Serratia sp. Tan611 strain was able to degrade n-alkanes (from C13 to C25). This bacterium was also shown to produce a biosurfactant, the emulsification index (E24) reaching 43.47% and 65.22%, against vegetable and crude oil, respectively. Finally, the formation of a biofilm was increased in the presence of crude oil. These observations make Serratia sp. Tan611 a good candidate for hydrocarbon bioremediation.

Aggregation and Amyloidogenicity of the Nuclear Coactivator Binding Domain of CREB-Binding Protein.

The nuclear coactivator binding domain (NCBD) of transcriptional co-regulator CREB-binding protein (CBP) is an example of conformationally malleable proteins that can bind to structurally unrelated protein targets and adopt distinct folds in the respective protein complexes. Here, we show that the folding landscape of NCBD contains an alternative pathway that results in protein aggregation and self-assembly into amyloid fibers. The initial steps of such protein misfolding are driven by intermolecular interactions of its N-terminal α-helix bringing multiple NCBD molecules into contact. These oligomers then undergo slow but progressive interconversion into ß-sheet-containing aggregates. To reveal the concealed aggregation potential of NCBD we used a chemically synthesized mirror-image d-NCBD form. The addition of d-NCBD promoted self-assembly into amyloid precipitates presumably due to formation of thermodynamically more stable racemic ß-sheet structures. The unexpected aggregation of NCBD needs to be taken into consideration given the multitude of protein-protein interactions and resulting biological functions mediated by CBP.

Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis-High Resolution Mass Spectrometry.

Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE-MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.

Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis - Mass spectrometry.

Significant growth of biopharmaceuticals requires powerful analytical methods to better understand their structure by establishing a complete characterization. To this end, a combination of bottom-up, middle-up and intact molecule levels with a capillary electrophoresis-mass spectrometry coupling has been performed to have a comprehensive picture of monoclonal antibodies. In this study, 7 worldwide health authorities approved mAbs have been analyzed to get information about their charge heterogeneity, the identification of post translational modifications (PTMs), their location and relative quantitation. Intact mAbs isoforms have been partially separated in less than 12 min and enabled to have a global illustration of mAbs heterogeneity and high masses PTMs characterization notably major N-glycosylation forms. Particularly, 2X-glycosylated and 1X-glycosylated forms have been partially separated. To deepen characterize PTMs carried by the backbone structure, advanced investigations at a middle-up level have been performed. Limited IdeS proteolysis allowed to study independently Fc/2 and F(ab)'2 fragments. Following the same separation conditions, isoforms of these fragments have been separated and data interpretation allowed to disclose additional PTMs as K-clip, oxidations or deamidations. A second intermediate level has been examined by adding a reduction step to establish a more precise assessment of PTMs and isoforms from the F(ab)'2 fragment. This reduction step released the light chains from the Fd fragment to get only 25 kDa fragments to analyze. CE-ESI-MS coupling allowed to get more information particularly about low masses PTMs. The precise location and relative quantitation of each PTM has been investigated at the peptidic level induced by a tryptic digestion of the studied mAbs. The concordance of the results shows the efficiency of the CE-ESI-MS coupling to characterize mAbs and highlight the need of the multi-level combination to get a comprehensive characterization of biotherapeutics.

Characterization of the Primary Structure of Cysteine-Linked Antibody-Drug Conjugates Using Capillary Electrophoresis with Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) enables the characterization of the primary structure of ADCs. An analytical method based on a derived bottom-up proteomic workflow is designed to provide detailed information about the amino acid sequence, the glycosylation profiling, and the location on the peptide backbone of the conjugated drugs. Here we describe the experimental protocol applied on the characterization of cysteine-linked brentuximab vedotin (Adcetris®).

Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016-2018.

Monoclonal antibodies (mAbs) and their related products as antibody-drug-conjugates (ADCs) or biosimilars represent a constantly growing class of molecules therapeutic proteins used as treatment against numerous diseases. These compounds can undergo several modifications which could alter the efficiency of treatments. In this context, several analytical methods were designed to deliver a comprehensive structural characterization and guarantee the quality of biotherapeutics. Capillary electrophoresis (CE) is considered today as a major technique for the analysis of biotherapeutics due to benefic characteristics as high resolution separation and miniaturized format. Different CE modes have been developed to characterize mAbs at different levels such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and capillary zone electrophoresis (CZE). Recent developments in CE-mass spectrometry (MS) coupling assessed this technology as a promising tool to obtain high level structural characterization of biopharmaceuticals. Moreover, upcoming techniques such as 2D CE-MS and microfluidic systems are now emerging to offer new possibilities beyond actual limits. This review will be dedicated to discuss the state-of-the-art CE-based methods for the characterization of mAbs and ADCs in the period 2016-2018.

Intact monoclonal antibodies separation and analysis by sheathless capillary electrophoresis-mass spectrometry.

Capillary electrophoresis-mass spectrometry coupling is a growing technique in biopharmaceutics characterization. Assessment of monoclonal antibodies is well known at middle-up and bottom-up levels to obtain information about the sequence, post-translational modifications and degradation products. Intact protein analysis is an actual challenge to be closer to the real protein structure. At this level, actual techniques are time consuming or cumbersome processes. In this work, a 20 minutes separation method has been developed to optimize characterization of intact monoclonal antibodies. Thus, separation has been done on a positively charged coated capillary with optimized volatile background electrolyte and sample buffer. Three world-wide health authorities approved monoclonal antibodies have been used to set up a rapid and ease of use method. Intact trastuzumab, rituximab and palivizumab isoforms have been partially separated with this method in less than 20 minutes under denaturing conditions. For each monoclonal antibody, 2X-glycosylated and 1X-glycosylated structures have been identified and separated. Concerning basic and acidic variants, potential aspartic acid isomerization modification and asparagine deamidation have been observed. Accurate mass determination for high-mass molecular species remains a challenge, but the progress in intact monoclonal antibodies separation appears very promising for biopharmaceutics characterization.

Revealing the potential of capillary electrophoresis/mass spectrometry: the tipping point.

The hyphenation of capillary electrophoresis and mass spectrometry (CE/MS) remains a minor technique compared with liquid chromatography/mass spectrometry (LC/MS), which represents nowadays the standard instrumentation, regardless of its introduction thirty years ago. However, from a theoretical point of view, CE coupling should be quite favorable especially with electrospray ionization mass spectrometry (ESI-MS). At the time, the sensitivity provided by CE/MS was often limited, due to hyphenation requirements, which at some point appeared to disqualify CE/MS from benefiting from the performance gain driving the evolution of MS instruments. However, this context has been significantly modified in a matter of a few years. The development of innovative CE/MS interfacing systems has enabled an important improvement regarding sensitivity and reinforced robustness in order to provide an instrumentation accessible to the largest scientific community. Because of the unique selectivity delivered by the electrophoretic separation, CE/MS has proved to be particularly relevant for the analysis of biological molecules. The conjunction of these aspects is motivating the interest in CE/MS analysis and shows that CE/MS is mature enough to enrich the toolbox of analytical techniques for the analysis of complex biological samples. Here we discuss the characteristics of the major types of high-sensitivity CE/ESI-MS instrumentation and emphasize the late evolution and future positioning of CE/MS analysis for the characterization of biological molecules like peptides and proteins, through some pertinent applications.

Entwined dimer formation from self-complementary bis-acridiniums.

A dicationic tweezer incorporating two acridinium moieties linked by a 2,6-diphenylpyridine spacer was shown to self-assemble in an entwined dimer both in acetonitrile and water. The reaction was studied according to solvent polarity, temperature and concentration conditions. The entwined structure was confirmed in the solid state via single-crystal X-ray diffraction.

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis - Mass spectrometry: Assessment and method validation.

Characterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision.

Omics for Precious Rare Biosamples: Characterization of Ancient Human Hair by a Proteomic Approach.

Omics technologies have far-reaching applications beyond clinical medicine. A case in point is the analysis of ancient hair samples. Indeed, hair is an important biological indicator that has become a material of choice in archeometry to study the ancient civilizations and their environment. Current characterization of ancient hair is based on elemental and structural analyses, but only few studies have focused on the molecular aspects of ancient hair proteins-keratins-and their conservation state. In such cases, applied extraction protocols require large amounts of raw hair, from 30 to 100 mg. In the present study, we report an optimized new proteomic approach to accurately identify archeological hair proteins, and assess their preservation state, while using a minimum of raw material. Testing and adaptation of three protocols and of nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) parameters were performed on modern hair. On the basis of mass spectrometry data quality, and of the required initial sample amount, the most promising workflow was selected and applied to an ancient archeological sample, dated to about 3880 years before present. Finally, and importantly, we were able to identify 11 ancient hair proteins and to visualize the preservation state of mummy's hair from only 500 µg of raw material. The results presented here pave the way for new insights into the understanding of hair protein alteration processes such as those due to aging and ecological exposures. This work could enable omics scientists to apply a proteomic approach to precious and rare samples, not only in the context of archeometrical studies but also for future applications that would require the use of very small amounts of sample.

Top-down and middle-down approach by fraction collection enrichment using off-line capillary electrophoresis - mass spectrometry coupling: Application to monoclonal antibody Fc/2 charge variants.

The characterization of complex protein mixtures represents one of the biggest challenge in many research fields such as biological or biopharmaceutical sciences. Out of all categories, monoclonal antibodies (mAbs) and related products drawn the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need for analytical methods to provide comprehensive in-depth characterization of these proteins. In this work, we developed a methodology using CE-UV/MALDI-MS to perform top-down or middle-down characterization after fraction collection enrichment applied to intact protein and mAbs samples. The performance of the method was evaluated with the rapid separation of three intact protein mixture. Good robustness of CZE separation and quality of MALDI-MS spectra and MALDI-ISD spectra of each protein confirms the usefulness of sample enrichment to obtain adequate quantity of deposed protein for top-down analysis and the proof of principle of the method. In a second step, the method was applied to the middle-down characterization of Fc/2 cetuximab variants. Identification of around 9% sequence coverage of Fc/2 cetuximab fragments allows to conclude on the feasibility of the strategy for middle-down characterization of Fc/2 cetuximab variants using CE-UV/MALDI-MS. Moreover, MALDI-ISD fragmentation of Fc/2 cetuximab variants confirm separation phenomenon based on the formation of Fc/2 dimers with and without C-terminal truncation.

Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products.

Out of all categories, monoclonal antibodies (mAbs), biosimilar, antibody-drug conjugates (ADCs) and Fc-fusion proteins attract the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide comprehensive in-depth characterization of these molecules. CE presents some obvious benefits as high resolution separation and miniaturized format to be widely applied to the analysis of biopharmaceuticals. CE is an effective method for the separation of proteins at different levels. capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) have been particularly relevant for the characterization of size and charge variants of intact and reduced mAbs, while CE-MS appears to be a promising analytical tool to assess the primary structure of mAbs and related products. This review will be dedicated to detail the current and state-of-the-art CE-based methods for the characterization of mAbs and related products.