Successive Multiple Ionic-Polymer Layer Coatings for Intact Protein Analysis by Capillary Zone Electrophoresis-Mass Spectrometry: Application to Hemoglobin Analysis.

Adsorption of analytes, e.g., proteins, often interfere with separation in CE, due to the relatively large surface of the narrow capillary. Coatings often are applied to prevent adsorption and to determine the electroosmotic flow (EOF), which is of major importance for the separation in CE. Successive multiple ionic-polymer layer (SMIL) coatings are frequently used for protein analysis in capillary electrophoresis resulting in high separation efficiency and repeatability. Here, the coating procedure of a five-layer SMIL coating is described using quaternized diethylaminoethyl dextran (DEAEDq) as polycation and poly(methacrylic acid) (PMA) as polyanion. Depending on the analyte, different polyions may be used to increase separation efficiency. However, the coating procedure remains the same.To demonstrate the applicability of SMIL coatings in CE-MS, human hemoglobin was measured in a BGE containing 2 M acetic acid. DEAEDq-PMA coating was found to be the most suitable for hemoglobin analysis due to relatively low reversed electroosmotic mobility leading to increased electrophoretic resolution of closely related proteoforms. Thereby, not only alpha and beta subunit of the hemoglobin could be separated, but also positional isoforms of glycated and carbamylated species were separated within 24 min.

Characterisation of a new online nanoLC-CZE-MS platform and application for the glycosylation profiling of alpha-1-acid glycoprotein.

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.

nanoCEasy: An Easy, Flexible, and Robust Nanoflow Sheath Liquid Capillary Electrophoresis-Mass Spectrometry Interface Based on 3D Printed Parts.

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.

Online top-down mass spectrometric identification of CE(SDS)-separated antibody fragments by two-dimensional capillary electrophoresis.

Size heterogeneity analysis by capillary sieving electrophoresis utilizing sodium dodecyl sulfate (CE(SDS)) with optical detection is a major method applied for release and stability testing of monoclonal antibodies (mAbs) in biopharmaceutical applications. Identification of mAb-fragments and impurities observed with CE(SDS) is of outstanding importance for the assessment of critical quality attributes and development of the analytical control system. Mass spectrometric (MS) detection is a powerful tool for protein identification and characterization. Unfortunately, CE(SDS) is incompatible with online MS-hyphenation due to strong ionization suppression of SDS and other separation buffer components. Here, we present a comprehensive platform for full characterization of individual CE(SDS)-separated peaks by CE(SDS)-capillary zone electrophoresis-top-down-MS. The peak of interest is transferred from the first to the second dimension via an 8-port valve to remove MS-incompatible components. Full characterization of mAb byproducts is performed by intact mass determination and fragmentation by electron transfer dissociation, higher-energy collisional dissociation, and ultraviolet photodissociation. This enables online determination of intact mass as well as sequence verification of individual CE(SDS)-separated peaks simultaneously. A more substantiated characterization of unknown CE(SDS) peaks by exact localization of modifications without prior digestion is facilitated. High sensitivity is demonstrated by successful mass and sequence verification of low abundant, unknown CE(SDS) peaks from two stressed mAb samples. Good fragmentation coverages are obtained by MS2, enabling unequivocal identification of these mAb-fragments. Also, the differentiation of reduced/non-reduced intra-protein disulfide bonds is demonstrated. In summary, a reliable and unambiguous online MS2 identification of unknown compounds of low-abundant individual CE(SDS) peaks is enabled.

Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics-both 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 ß-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 ß-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 ß-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.

Classroom-Based Micro-Sessions of Functional High-Intensity Circuit Training Enhances Functional Strength but Not Cardiorespiratory Fitness in School Children-A Feasibility Study.

The present study assessed the short-term effect of 6 min classroom-based micro-sessions of multi-joint functional high-intensity circuit training (FunctionalHIIT) performed by students during regular classes on parameters related to functional strength and cardiorespiratory fitness. In this randomized controlled 4-week study, 17 students (11 male; 6 female; age: 11.6 ± 0.2 years) performed 6 min of FunctionalHIIT (targeting >17 on the Borg scale) 4 days per week during regular school classes and 18 students (11 male; 7 female; age: 11.7 ± 0.3 years) served as control group (CG) without any additional in-class physical activity. The FunctionalHIIT group completed 86% of all planned sessions (mean duration: 6.0 ± 1.5 min) with a mean RPE of 17.3 ± 2.1. Body height, mass and BMI did not differ between the groups at baseline or between pre- and post-testing (p > 0.05; eta2 ≤ 0.218). The performances in lateral jumping (p < 0.000; part eta2 = 0.382; Δ% 4.6 ± 8.6), sit-ups (p < 0.000; part eta2 = 0.485; Δ% 3.1 ± 8.6) and 20-m sprints (p < 0.000; part eta2 = 0.691; Δ% 15.8 ± 5.4) improved in both groups with greater increase following FunctionalHIIT. No baseline differences and no interaction effects occurred in performance of 6 min run, flexibility, push-ups, balance, and long jump. Classroom-based FunctionalHIIT sessions, performed 4 days per week during 4 weeks did not improve variables related to aerobic endurance performance but enhanced certain parameters of functional strength in schoolchildren. As time is limited in the educational system of schools, FunctionalHIIT during regular school classes could offer a new perspective for increasing functional strength in schoolchildren.

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.