Electrophoretic characterization of LNP/AAV-encapsulated nucleic acids: Strengths and weaknesses.

The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs (>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.

Two capillary approach for a multifunctional nanoflow sheath liquid interface for capillary electrophoresis-mass spectrometry.

CE hyphenated to ESI-MS (CE-ESI-MS) is a well-established technique to analyze charged analytes in complex samples. Although various interfaces for CE-MS coupling are commercially available, the development of alternatives which combine sensitivity, simplicity, and robustness remains a topic of research. In this work, a nanoflow sheath liquid CE-MS interface with two movable capillaries inside a glass emitter is described. The setup enables a separation mode and a conditioning mode to guide the separation capillary effluent either into the electrospray or to the waste, respectively. This enables to exclude parts of the analysis from MS detection and unwanted matrix components reaching the mass spectrometer, comparable to divert valves in LC-MS coupling. Also, this function improves the overall robustness of the system by reduction of particles blocking the emitter. Preconditioning with electrospray interfering substances and even the application of coating materials for every analysis is enabled, even while the separation capillary is built into the interface with running electrospray. The functionality is demonstrated by analyses of heavy matrix bioreactor samples. Overall, this innovation offers a more convenient installation of the interface, improved handling with an extended lifetime of the emitter tips and additional functions compared to previous approaches, while keeping the higher sensitivity of nanoflow CE-MS-coupling.

12-Oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), a novel glutathione-containing eicosanoid generated via the 12-lipoxygenase pathway in human platelets.

Biologically active glutathione (GSH) conjugates of oxygenated fatty acids comprise a group of pro- and anti-inflammatory lipid mediators. While arachidonic acid (AA)-derived conjugates, as the cysteinyl leukotrienes (cys-LTs) and eoxins (EXs) have pro-inflammatory properties, conjugates in tissue regeneration (CTRs) biosynthesized from docosahexaenoic acid (DHA) exhibit pro-resolving activity. Human platelets express abundant amounts of platelet-type 12-lipoxygenase (pt12-LOX) and leukotriene C4 synthase (LTC4S). However, the only two described GSH conjugates formed by platelets are the AA-derived cys-LTs and the recently reported maresin CTRs (MCTRs). While cys-LTs are biosynthesized in a transcellular mechanism via the action of 5-LOX and LTC4S, MCTR1 is formed by 12-LOX and a yet unidentified GSH S-transferase (GST). Here, we present a novel GSH conjugate formed from AA via the 12-LOX pathway in human platelets. The 12-oxo-glutathione adduct, 12-oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), was identified by mass spectrometry using positive electrospray ionization. The structural proposal is supported by fragmentation data of the labeled metabolite obtained after incubation of deuterated AA (AA-d8). In platelets as well as in HEK293 cells stably expressing pt12-LOX, TOG10 biosynthesis was inhibited by the 12-LOX inhibitor ML-355 (5 µM), which confirms the involvement of pt12-LOX. Interestingly, TOG10 was formed independently of LTC4S in platelets. This is in accordance with the observation that the conjugate was also generated by AA-stimulated HEK_12-LOX cells in absence of LTC4S. Nevertheless, TOG10 can also be formed by LTC4S as the biosynthesis in HEK_12-LOX_LTC4S cells was reduced by the specific LTC4S inhibitor TK04a. In summary, TOG10 was identified as a new AA-derived GSH conjugate generated in human platelets via the action of pt12-LOX in combination with a GST.

Enrichment-free analysis of anionic micropollutants in the sub-ppb range in drinking water by capillary electrophoresis-high resolution mass spectrometry.

Reversed-phase liquid chromatography (RPLC) used for water analysis is not ideal for the analysis of highly polar and ionic contaminants because of low retention. Capillary electrophoresis (CE), on the other hand, is perfectly suited for the separation of ionic compounds but rarely applied in environmental analysis due to the weak concentration sensitivity when coupled to mass spectrometry (MS). However, novel interface designs and MS technology strongly improve the sensitivity. Here, a method is presented enabling the screening of anionic micropollutants in drinking water without sample pretreatment by coupling of CE to an Orbitrap mass spectrometer by a nanoflow sheath liquid interface. Targeted analysis of halogenated acetic acids, trifluoromethanesulfonic acid, and perfluorooctanoic and perfluorooctanesulfonic acid was conducted in drinking water samples which were chlorinated for disinfection. A bare fused silica capillary with an optimized background electrolyte (BGE) for separation consisting of 10% acetic acid with 10% isopropanol with large volume sample injection and optimized interface parameters offer limits of quantification in the range of < 0.1 to 0.5 µg/L with good linearity (R2 > 0.993) and repeatability (14% standard deviation in area). Concentrations of the target analytes ranged from 0.1 to 6.2 µg/L in the water samples. Masses corresponding to halogenated methanesulfonic acids have been found as suspects and were subsequently verified by standards. Mono-, dichloro-, and bromochloro methanesulfonic acid were quantified in a range of 0.2 to 3.6 µg/L. Furthermore, five sulfonic acids, four organosulfates, and the artificial sweeteners acesulfame and cyclamate as well as inorganics such as halides, halogenates, phosphate, and sulfate could be determined as suspects among more than 300 features in a non-targeted screening. Overall, this approach demonstrates the great potential of CE-nanoESI-MS for the screening of ionic contaminants in environmental samples, complementary to chromatographic approaches.

Recent advances in capillary electrophoresis-mass spectrometry: Instrumentation, methodology and applications.

Capillary electrophoresis (CE) offers fast and high-resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user-friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano-electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE-MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two-dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE-modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.

Characterization of a nanoflow sheath liquid interface and comparison to a sheath liquid and a sheathless porous-tip interface for CE-ESI-MS in positive and negative ionization.

Coupling of capillary electrophoresis to electrospray mass spectrometry still remains challenging and a topic of research to find the best interface regarding sensitivity, robustness, and ease of use. Here, a nanoflow sheath liquid interface for CE-ESI-MS is presented and compared to both a standard triple-tube sheath liquid and a porous-tip sheathless interface for three groups of analytes. The nanoflow sheath liquid interface with a separation capillary inserted into a glass emitter was initially characterized to facilitate optimization and method development. Implementation of a shut-off valve, syringe pump, and inline filter enabled easy handling and fast analyses, repeatable both in positive and negative modes (intra-day RSD of 6.6 to 12.0%). The same setup was used for sheathless interfacing by exchanging the emitter and using a porous etched tip separation capillary. Both nanoflow interfaces showed similar performance. Average peak areas using the nanoflow sheath liquid interface were a factor of 38 for 6 organic acids in negative mode, 114 and 36 for the light and heavy chain of a monoclonal antibody, and 13 higher for peptides in positive mode compared to the triple-tube interface. This first direct comparison of the three most common interfaces exhibits a strong improvement in sensitivity to the same extent for both nanoflow interfaces, where sheath liquid interfaces offer full flexibility in method development.

Non-targeted LC-MS and CE-MS for biomarker discovery in bioreactors: Influence of separation, mass spectrometry and data processing tools.

Liquid separation coupled to mass spectrometry is often used for non-targeted analyses in various fields, such as metabolomics. However, the combination of non-standardized methods, various mass spectrometers (MS) and processing tools for data evaluation affect biomarker discovery potentially. Here, we present a comprehensive study of these factors based on non-targeted liquid chromatography coupled to time-of-flight (TOF) and Orbitrap MS and capillary zone electrophoresis to Orbitrap analyses of the same bioreactor samples, describing the correlation of its gas yield with changing feature signal intensity. The three datasets were processed with MZmine 2 and XCMS online and subsequential Partial Least Square Regression (PLSR) with Variable Importance in Projection (VIP) ranking for feature prioritization. The six feature tables were compared to evaluate their overlap of shared features and the influence of the processing software and MS instrument on the VIP values and fold changes. The overlaps, defined as a fraction of one feature table found in the comparative table, were from 27% to 57% for the comparison of MZmine and XCMS and from 15% to 50% between Orbitrap and TOF data sets, respectively. Considering the most relevant features only (VIP >1.5), the overlaps were increased significantly in all cases from 26% to 95%. For the same data set, both VIP values and fold changes were well correlated, however, varied significantly between TOF and Orbitrap. CE-MS showed higher total feature numbers compared to LC-MS, most likely due to its more appropriate selectivity, different sample preparation, and/or the sensitive nano-ESI interface. Since only less than 10% of MS/MS data overlapped, CE-MS provided complementary information to LC-MS. Overall, our systematic study proves the benefits of using different separation techniques and processing tools but also indicates a significant influence of mass spectrometry on comprehensive biomarker discovery.