Optimisation of the separation of therapeutic phosphorothioate oligonucleotides by hydrophilic interaction liquid chromatography and capillary zone electrophoresis.

Introduction: Therapeutic oligonucleotides are short nucleic acids chemically synthetized that play a major role in gene regulation and the treatment of various diseases. They can target DNA, RNA, proteins, posttranslational protein modifications, carbohydrates, lipids or metabolites. Oligonucleotides are easily in-vivo degraded and need to be modified to improve their pharmacokinetic and pharmacodynamics properties. Phosphorothioate oligonucleotides (PS ON) is the most dominating modification where the oxygen atom of the phosphodiester bond is replaced by a sulfur atom. This result in enhanced resistance against nucleases degradation and thus increased half-life. Goals: Since many therapeutic oligonucleotides are arriving on the global market, there is an important need for appropriate analytical techniques to ensure their quality control. In this work, we optimized hydrophilic interaction liquid chromatography and capillary zone electrophoresis methods to detect PS ON impurities. Material and methods: Two complex mixtures were used to optimize the separation methods. Firstly, a mixture containing short PS ON of identical sequence varying by the position of the phosphodiester bonds will be analyzed. Secondly, a mixture of 20 PS ON of different lengths and thus different number of PS linkages will be used. Results and discussions: In hydrophilic interaction liquid chromatography, the stationary phase composition and the mobile phase composition and gradient were carefully optimized. In capillary zone electrophoresis, the pH and molarity of the background electrolyte will be studied. The final developed methods were compared in terms of peak efficiency, resolution and analysis time. The advantages and disadvantages related to each technique will be discussed. Conclusions: We demonstrated that hydrophilic interaction liquid chromatography and capillary electrophoresis are suitable techniques to differentiate closely related PS ON and could easily be applied for the quality control of those emerging medicines

CE-MS/MS for bottom-up proteomics: comparaison of two coupling interfaces with ion mobility qTOF

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein mixtures. As the samples are of high complexity and that some proteins can be at very low concentrations, efficient and sensitive instruments have to be used in order to maximize peptide identification. Nowadays, capillary electrophoresis tandem mass spectrometry (CE-MS/MS) has gained interest in proteomic analysis as it is considered as complementary to the gold standard method, namely reverse phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS). However, the coupling of CE with MS is not straightforward. Indeed robust interface is needed in order to conserve the high-resolution in-capillary separation while ensuring a stable spray. Among the commercialized interfaces, the coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) have been tested for the analysis of BSA and E. coli proteome digests. Both interfaces were coupled with an IMS-qTOF-MS. Several parameters were optimized in order to maximize the sensitivity, such as the composition of the sheath liquid and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). In our study, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. At the end, spray stability was found as the main strength of the triple tube interface, whereas the EMASS-II interface was found to provide higher sensitivity thanks to the reduced flow rate of the sheath liquid

New role for Emerin, a key inner nuclear membrane protein, as an enhancer of the autophagosome formation in the C16-ceramide autophagy pathway.

To date, precise roles of EMD remain poorly described. In this paper, we investigate the role of EMD in the C16-ceramide autophagy pathway. Ceramides are bioactive signalling molecules acting notably in the regulation of cell growth, differentiation or cell death. However, the mechanisms by which they mediate these pathways are not fully understood. We found that C16-ceramide induces EMD phosphorylation on its LEM domain through PRKACA. Upon ceramide treatment, phosphorylated EMD binds LC3 leading to an increase of the autophagosomes formation. These data suggest a new role of EMD as an enhancer of autophagosomes formation in the C16-ceramide autophagy pathway in colon cancer cells

Proteomic analysis using capillary electrophoresis hyphenated with high resolution mass spectrometry: comparison of two coupling interfaces

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC-MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. The use of dynamic pH junction allowed the identification of more peptides and proteins compared to conventional injections. The sheath liquid composition was also optimized in order to enhance signal intensity. A nanoflow sheath liquid interface (EMASS-II) was compared to the traditional coaxial sheath liquid interface (Triple tube) in terms of number of identified peptides and proteins as well as in terms of sensitivity (peak area and peak height). The use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by a factor of 4 and 6-fold, respectively, compared to the Triple tube

Capillary electrophoresis tandem mass spectrometry for proteomic analysis: optimization and comparison of two coupling interfaces

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new proteic entities. As the samples are of high complexity and that some proteins could be present at very low concentrations, efficient and sensitive instruments have to be used in order to maximize peptide identification. Nowadays, capillary electrophoresis tandem mass spectrometry (CE-MS/MS) has gained interest in proteomic analysis as it is considered as complementary to the gold standard method, namely reverse phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS). However, the coupling of CE with MS is not straightforward. Indeed, robust interface is needed in order to conserve the high-resolution in-capillary separation while ensuring a stable spray. For this purpose, optimization of basic parameters such as BGE composition was first carried out using a simple peptide mix. Several parameters were then optimized in order to maximize the sensitivity, such as the composition of the sheath liquid, the interface position and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). Finally, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. In our study, two commercialized interfaces were compared by analysing E. coli proteome digest. The coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) were both coupled with an IMS-qTOF-MS. Eventually, spray stability was found to be the main strength of the triple tube interface, whereas the EMASS-II interface was found to provide higher sensitivity thanks to the reduced flow rate of the sheath liquid

Hyphenation of capillary zone electrophoresis with mass spectrometry for proteomic analysis: Optimization and comparison of two coupling interfaces

Capillary electrophoresis tandem mass spectrometry (CE–MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC–MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NH4Ac) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity. A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE–MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest. © 202

Ion mobility high resolution mass spectrometry coupled to HILIC and CZE for the characterization of phosphodiester and phosphorothioate oligonucleotides

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. With the emergence of oligonucleotides on the market, it is of increasing importance to develop analytical methods to study those modified oligonucleotides and their impurities. Chromatographic and electrophoretic approaches may be used for that purpose. In this work, poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS) were studied. For the chromatographic approach, Hydrophilic Interaction Liquid Chromatography (HILIC) was chosen as it represents good alternative to commonly used ion-pair reversed-phase liquid chromatography for the analysis of polar compounds. Moreover, it avoids the use of ion pairing agents, which makes it more compatible with mass spectrometric detection. For the electrophoretic approaches, a capillary zone electrophoresis (CZE) with a MS-compatible background electrolyte was employed. Both techniques were coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides