The ever-increasing complexity of biological samples to be analysed by mass spectrometry has led to the necessity of sophisticated separation techniques, including multidimensional separation. ...Despite a high degree of orthogonality, the coupling of liquid chromatography (LC) and capillary zone electrophoresis (CZE) has not gained notable attention in research. Here, we present a heart-cut nanoLC-CZE-ESI-MS platform to analyse intact proteins. NanoLC and CZE-MS are coupled using a four-port valve with an internal nanoliter loop. NanoLC and CZE-MS conditions were optimised independently to find ideal conditions for the combined setup. The valve setup enables an ideal transfer efficiency between the dimensions while maintaining good separation conditions in both dimensions. Due to the higher loadability, the nanoLC-CZE-MS setup exhibits a 280-fold increased concentration sensitivity compared to CZE-MS. The platform was used to characterise intact human alpha-1-acid glycoprotein (AGP), an extremely heterogeneous N-glycosylated protein. With the nanoLC-CZE-MS approach, 368 glycoforms can be assigned at a concentration of 50 μg/mL as opposed to the assignment of only 186 glycoforms from 1 mg/mL by CZE-MS. Additionally, we demonstrate that glycosylation profiling is accessible for dried blood spot analysis (25 μg/mL AGP spiked), indicating the general applicability of our setup to biological matrices. The combination of high sensitivity and orthogonal selectivity in both dimensions makes the here-presented nanoLC-CZE-MS approach capable of detailed characterisation of intact proteins and their proteoforms from complex biological samples and in physiologically relevant concentrations.
Graphical abstract
Capillary electrophoresis-mass spectrometry (CE-MS) is a powerful tool in various fields including proteomics, metabolomics, and biopharmaceutical and environmental analysis. Nanoflow sheath liquid ...(SL) CE-MS interfaces provide sensitive ionization, required in these fields, but are still limited to a few research laboratories as handling is difficult and expertise is necessary. Here, we introduce nanoCEasy, a novel nanoflow SL interface based on 3D printed parts, including our previously reported two capillary approach. The customized plug-and-play design enables the introduction of capillaries and an emitter without any fittings in less than a minute. The transparency of the polymer enables visual inspection of the liquid flow inside the interface. Robust operation was systematically demonstrated regarding the electrospray voltage, the distance between the emitter and MS orifice, the distance between the separation capillary and emitter tip, and different individual emitters of the same type. For the first time, we evaluated the influence of high electroosmotic flow (EOF) separation conditions on a nanoflow SL interface. A high flow from the separation capillary can be outbalanced by increasing the electrospray voltage, leading to an overall increased electrospray flow, which enables stable operation under high-EOF conditions. Overall, the nanoCEasy interface allows easy, sensitive, and robust coupling of CE-MS. We aspire the use of this sensitive, easy-to-use interface in large-scale studies and by nonexperts.
Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control of therapeutic monoclonal antibodies (mAbs). The highly selective and commonly ...applied capillary zone electrophoresis (CZE) method containing high amounts of ε-aminocaproic acid (EACA) provides a detailed and robust charge heterogeneity profile of intact mAb variants. Nevertheless, the exact location of protein modifications within these charge profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) is a promising tool for this purpose; however, EACA is incompatible with electrospray. In this context, we present a two-dimensional CZE-CZE-MS system to combine efficient charge variant separation of intact mAbs with subsequent peptide analysis after in-capillary digestion of selected charge variants. The first dimension is based on a generic CZE(EACA) method in a fused silica capillary. In the second dimension, a neutral-coated capillary is used for in-capillary reduction and digestion with Tris(2-carboxyethyl)phosphine (TCEP) and pepsin, followed by CZE separation and MS/MS-characterization of the resulting peptides. The setup is demonstrated using stressed and nonstressed mAbs where peaks of basic, main, and acidic variants were transferred in a heart-cut fashion, digested, and characterized on the peptide level. Sequence coverages of more than 90% were obtained for heavy chain (HC) and light chain (LC) for four different mAbs, including low-abundance variants (<2% of the main peak). Frequently observed modifications (deamidation, oxidation, etc.) could be detected and localized. This study demonstrates a proof-of-concept for identification and localization of protein modifications from CZE charge heterogeneity profiles and, in this way, is expected to support the development and quality control testing of protein pharmaceuticals.
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.
•Combination of dispersive Raman spectroscopy and principal component analysis.•Systematic study of benefits and limits for the characterization of counterfeits.•Distinction between different ...coatings, amount of API and excipients is possible.•Systematic study of storage of ASS+C tablets at five different conditions.•It is possible to detect chemical changes well before visual changes appear.
In the last decade, counterfeit pharmaceutical products have become a widespread issue for public health. Raman spectroscopy which is easy, non-destructive and information-rich is particularly suitable as screening method for fast characterization of chemicals and pharmaceuticals. Combined with chemometric techniques, it provides a powerful tool for the analysis and determination of counterfeit medicines. Here, for the first time, a systematic study of the benefits and limitations of Raman spectroscopy for the analysis of pharmaceutical samples on a large set of model tablets, varying with respect to chemical and physical properties, was performed. To discriminate between the different mixtures, a combination of dispersive Raman spectroscopy performing in backscattering mode and principal component analysis was used. The discrimination between samples with different coatings, a varying amount of active pharmaceutical ingredients and a diversity of excipients were possible. However, it was not possible to distinguish between variations of the press power, mixing quality and granulation. As a showcase, the change in Raman signals of commercial acetylsalicylic acid effervescent tablets due to five different storage conditions was monitored. It was possible to detect early small chemical changes caused by inappropriate storage conditions. These results demonstrate that Raman spectroscopy combined with multivariate data analysis provides a powerful methodology for the fast and easy characterization of genuine and counterfeit medicines.
Hemoglobin (Hb) constitutes an important protein in clinical diagnosticsboth in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical ...modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and β-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and β-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the β-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.
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 (
R
2
> 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.
Successive multiple ionic-polymer layers (SMIL) coatings have been often used in capillary electrophoresis due to their simplicity to implement and regenerate. However, the performances of the ...separation are strongly dependent on the nature of the polyelectrolyte partners used to build the SMIL coating. In this work, we investigate new couples of polyelectrolytes that were not tested before: namely, polybrene (PB), quaternized diethylaminoethyl dextran (DEAEDq) and ε-poly(lysine) (ε-PLL), as polycations, in combination with poly(acrylic acid), dextran sulfate, poly(styrenesulfonate), poly(methacrylic acid) and poly(l-lysine citramide), as polyanions. Systematic study of intra- and inter-capillaries repeatabilities/reproducibilities was performed based on the determination of migration time, separation efficiency and electroosmotic mobility. Interestingly, the electroosmotic flow was found to vary with the nature of the polycation on a broad range of electroosmotic mobility decreasing in magnitude in the order of PB>ε-PLL > DEAEDq, whatever the polyanion associated. Application of the coatings to the separation of proteins is illustrated in a 0.5 M acetic acid BGE, including CE-MS separation of ribonuclease B-glycoforms of the same mass (positional or structural isomers).
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•5-layers successive polyelectrolyte coating used for protein analysis in CE.•Electroosmotic mobility varies from −43 to −63 Tiselius units depending on polycation.•Coatings with poly(l-lysine citramide) gave the best separation efficiencies.•Average RSD on migration times (resp. separation efficiency) was 1.7% (resp. 9.6%).•CE-MS separation of positional isomers of the glycoprotein ribonuclease B.
The examination of protein glycosylation is of high importance, especially in the (bio)pharmaceutical sector. The analysis of protein glycosylation is conducted routinely in high performance by ...capillary electrophoresis with laser-induced fluorescence (CE/LIF) using 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans. In this work we present an optimized capillary electrophoresis/time-of-flight mass spectrometry (CE/TOF-MS) methodology for these labeled glycans, which combines the high separation performance of CE with the high resolution, accuracy, and speed of TOF-MS for eased glycan identification. The system based on an acidic background electrolyte (BGE) provides a migration direction analogue to routine CE/LIF systems. Different BGE compositions, capillary dimensions, coatings, and instrumental parameters were tested to optimize the system with respect to separation efficiency and robustness. Subsequently, the CE/MS method optimized for acidic conditions was compared to an alkaline CE/MS method. Further, the mobilities of six APTS-labeled complex-type N-glycans were compared for both CE/MS methods and two standard CE/LIF approaches. For the acidic and alkaline BGE systems, the mobilities of sialylated glycans were shifted relative to nonsialylated glycans in comparison to common CE/LIF systems. However, in this study a straightforward unequivocal peak assignment was achieved for all unknown glycans in a medium complex glycan mixture from a fusion protein.
Advanced analytical techniques play a crucial role in the pharmaceutical and biotechnological field. In this context, capillary electrophoresis/mass spectrometry (CE/MS) has attracted attention due ...to efficient and selective separation in combination with powerful detection allowing identification and detailed characterization. Method developments and applications of CE/MS have been focused on questions not easily accessible by liquid chromatography/mass spectrometry (LC/MS) as the analysis of intact proteins, carbohydrates, and various small molecules, including peptides. Here, recent approaches and applications of CE/MS relevant to (bio)pharmaceuticals are reviewed and discussed to show actual developments and future prospects. Based on other reviews on related subjects covering large parts of previous works, the paper is focused on general ideas and contributions of the last 2 years; for the analysis of glycans, the period is extended back to 2006.
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