Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird\'s-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

A capillary zone electrophoresis (CZE) system was coupled to an Orbitrap mass spectrometer operating in a data-independent acquisition (DIA) mode for in-depth proteomics analysis. The performance of this CZE-DIA-MS system was systemically evaluated and optimized under different operating conditions. The performance of the fully optimized CZE-DIA-MS system was subsequently compared to the one by using the same CZE-MS system operating in a data-dependent acquisition (DDA) mode. The experimental results show that the numbers of identified peptides and proteins acquired in the DIA mode are much higher than the ones acquired in the DDA mode, especially with the small sample loading amount. Specifically, the numbers of identified peptides and proteins acquired in the DIA mode are 1.8-fold and 2-fold higher than the ones acquired in the DDA mode by using 12.5 ng Hela digests. The proteins identified in the DIA mode also cover almost all the proteins identified in the DDA mode. In addition, a potential cancer biomarker protein, carbohydrate antigen 125, undetected in the DDA mode, can be easily identified in the DIA mode even with 12.5 ng Hela digests. The performance of the CZE-DIA-MS system for in-depth proteomics analysis with a limited sample amount has been fully demonstrated for the first time through this study.

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 μg of cell lysate as the starting material. Three thousand seven hundred ninety-six (N = 2, RSD = 4.95%) protein groups and 20,577 (N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.

BACKGROUND: 2,6-Disubstituted piperidin-3-ols are an important group of piperidine alkaloids found in species such as Senna spectabilis, whose main constituents include cassine and spectaline, compounds with relevant pharmacological activity. The analysis of these compounds is challenging due to the complexity of plant extracts and the absence of chromophores capable of absorbing ultraviolet (UV) radiation.
OBJECTIVE: This paper presents a new analytical method to separate and quantify the non-UV-absorbing alkaloids present in ethanol extracts from S. spectabilis flowers using capillary zone electrophoresis (CZE) with indirect UV detection.
METHODS: The optimized CZE method employs a background electrolyte containing 60 mM histidine (His), 15 mM α-cyclodextrin, 20% acetonitrile (ACN), and pH-adjusted to 4.7 with acetic acid (AcOH).
RESULTS: The limit of detection (LOD) values was 10.2 and 13.9 mg L-1 for cassine and spectaline, respectively. For both analytes, the precision data were better than 2% of relative standard deviation (RSD) for migration times and peak areas. To evaluate the applicability of the developed method, ethanolic extracts from S. spectabilis flowers were prepared and analyzed.
CONCLUSIONS: Thereby, the method proved to be efficient and complementary to conventional techniques, offering a cost-effective alternative in the quantification of the non-UV-absorbing piperidine alkaloids present in plant extracts.

Therapeutic peptides have emerged as an innovative and promising class of therapeutic compounds in modern medicine. Synthetic peptide analogs triptorelin and lanreotide are known for their pronounced clinical versatility and potency. In this study, we present the development and validation of novel methods based on capillary zone electrophoresis performed in hydrodynamically closed system (HCS) and paired with ultraviolet detection and repeated injection sample introduction. To the best of our knowledge, we developed the first capillary electrophoresis-based method for the determination of lanreotide, and concurrently, the first HCS method for the determination of triptorelin. Maximal separation efficiency and signal intensity were achieved using background electrolytes composed of 50 mM formic acid with the addition of 0.05% (v/v) methyl-hydroxyethyl cellulose. The proposed methods exhibit favorable performance characteristics, namely, calibration curve (r2 exceeding 0.99), low limits of detection (0.25 µg/mL in a water matrix and 0.5 µg/mL in synthetic urine), acceptable precision (relative standard deviation ranging from 2.2% to 9.6% for intraday repeatability and between 5.2% and 14.9% for interday reproducibility), and accuracy (relative errors falling within the 91.1%-107.8% range). The method for triptorelin determination was then used for its quantification in a commercially available drug dosage form (powder for injection) and in spiked synthetic urine samples. The developed methods were also evaluated according to the novel blue applicability grade index, revealing their superior applicability. The results collectively point out the potential of the proposed methods for both quality control and clinical investigations.

Electrophoresis is a useful diagnostic tool for detecting inflammation, including inflammation associated with infectious diseases (eg, aspergillosis in penguins). To our knowledge, reference intervals are not available for plasma proteins via electrophoresis in Humboldt penguins (Spheniscus humboldti). Therefore, preliminary reference intervals for blood plasma proteins measured by capillary zone electrophoresis were calculated for Humboldt penguins from a single zoological collection, and possible differences between the sexes and the ages of the birds were evaluated. Lithium heparinized plasma samples from 39 Humboldt penguins were analyzed. The following sex- and age-independent reference intervals were calculated: total protein 33.8-70.4 g/L, prealbumin 1.9-4.9 g/L, albumin 12.9-31.1 g/L, albumin: globulin ratio 0.7-1.7, α-globulins 4.5-11.6 g/L, β-globulins 5.6-20.6 g/L, and γ-globulins 2.6-8.4 g/L. Male penguins had a significantly (P = 0.047) higher albumin: globulin ratio and lower percentage of β-globulins (P = 0.015) in comparison with female penguins. Prealbumin (g/L) significantly (P = 0.021) decreased with increased age of the penguins. These results showed some differences between the sexes and ages of the penguins, which should be considered when interpreting the results. Further studies are needed to determine whether differences in other age groups or seasons exist, and also to evaluate which infectious diseases affect plasma proteins and how the reference values calculated here may deviate in ill penguins.

Monitoring plasma concentrations of β-lactam antibiotics is crucial, particularly in critically ill patients, where variations in concentrations can lead to treatment failure or adverse events. Standardized antimicrobial regimens may not be effective for all patients, especially in special groups with altered physiological parameters. Pharmacokinetic/pharmacodynamic (PK/PD) studies highlight the time-dependent antibacterial activity of these antibiotics, emphasizing the need for personalized dosing. Therapeutic drug monitoring (TDM) is essential, requiring rapid and accurate analytical methods for precise determination of drugs in biological material (typically plasma or serum). This study presents a novel capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) method designed for the simultaneous quantification of five penicillin antibiotics, two cephalosporins, one carbapenem, and two β-lactamase inhibitors in a single run. The method involves a simple sample pretreatment-precipitation with organic solvent-and has a run time of 20 min. Optimization of CZE separation conditions revealed that 20 mM ammonium hydrogen carbonate (NH4HCO3) serves as the optimal background electrolyte (BGE). Positive electrospray ionization (ESI) mode, with isopropyl alcohol (IP)/10 mM ammonium formate water solution (50/50, v/v) as the sheath liquid, was identified as the optimal condition for MS detection. Method validation according to the Food and Drug Administration (FDA) guideline for development of bioanalytical methods demonstrated satisfactory selectivity, linearity, recovery, robustness, and stability. The method\'s practicality was evaluated using the Blue Applicability Grade Index (BAGI), yielding a score of 77.5. Moreover, the greenness of the proposed method was evaluated by two commonly used metric tools-Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI). The developed CZE-MS/MS method offers a practical and reliable approach for quantifying a broad spectrum of β-lactam antibiotics in plasma. Its ability to simultaneously quantify multiple analytes in a single run, coupled with a straightforward sample pretreatment, positions it as a valuable and prospective tool for TDM in critically ill patients.

OBJECTIVE: Voxelotor can increase hemoglobin levels in patients living with sickle cell disease (SCD). A clinician who is monitoring voxelotor response may want to know whole-blood voxelotor concentration, but this cannot be measured in most clinical settings. However, voxelotor has been demonstrated to cause \"peak splitting\" in common methods of hemoglobin measurement such as capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC). We hypothesized that we could use the size of the peak split to estimate the whole-blood concentration.
METHODS: Blood from people with SCD was dosed with known concentrations of voxelotor, and multiparameter regression was used to derive the relationship of voxelotor concentration to the degree of peak splitting observed. To validate these equations, 21 patients started on voxelotor at 1500 mg/d had blood samples drawn at days 0, 14, 30, and 60. Samples were sent out for gold standard voxelotor concentration testing. The derived equations were then used to calculate voxelotor concentration.
RESULTS: Calculated concentrations correlated strongly with measured concentrations for both CZE (R2 = 0.83, P < .001) and HPLC (R2 = 0.76, P < .001). Voxelotor concentration also had a significant effect on increases in hemoglobin (R2 = 0.40, P < .001).
CONCLUSIONS: Thus, peak splitting CZE and HPLC can be used to estimate voxelotor concentration.

Messenger RNA (mRNA) vaccines represent a landmark in vaccinology, especially with their success in COVID-19 vaccines, which have shown great promise for future vaccine development and disease prevention. As a platform technology, synthetic mRNA can be produced with high fidelity using in vitro transcription (IVT). Magnesium plays a vital role in the IVT process, facilitating the phosphodiester bond formation between adjacent nucleotides and ensuring accurate transcription to produce high-quality mRNA. The development of the IVT process has prompted key inquiries about in-process characterization of magnesium ion (Mg++) consumption, relating to the RNA polymerase (RNAP) activation, fed-batch mode production yield, and mRNA quality. Hence, it becomes crucial to monitor the free Mg++ concentration throughout the IVT process. However, no free Mg++ analysis method has been reported for complex IVT reactions. Here we report a robust capillary zone electrophoresis (CZE) method with indirect UV detection. The assay allows accurate quantitation of free Mg++ for the complex IVT reaction where it is essential to preserve IVT samples in their native-like state during analysis to avoid dissociation of bound Mg complexes. By applying this CZE method, the relationships between free Mg++ concentration, the mRNA yield, and dsRNA impurity level were investigated. Such mechanistic understanding facilitates informed decisions regarding the quantity and timing of feeding starting materials to increase the yield. Furthermore, this approach can serve as a platform method for analyzing the free Mg++ in complex sample matrices where preserving the native-like state of Mg++ binding is key for accurate quantitation.

Mass spectrometry (MS)-based top-down proteomics (TDP) has revolutionized biological research by measuring intact proteoforms in cells, tissues, and biofluids. Capillary zone electrophoresis-tandem MS (CZE-MS/MS) is a valuable technique for TDP, offering a high peak capacity and sensitivity for proteoform separation and detection. However, the long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which is a crucial aspect for large-scale studies. This work investigated the long-term qualitative and quantitative reproducibility of CZE-MS/MS for TDP for the first time, focusing on a yeast cell lysate. Over 1000 proteoforms were identified per run across 62 runs using one linear polyacrylamide (LPA)-coated separation capillary, highlighting the robustness of the CZE-MS/MS technique. However, substantial decreases in proteoform intensity and identification were observed after some initial runs due to proteoform adsorption onto the capillary inner wall. To address this issue, we developed an efficient capillary cleanup procedure using diluted ammonium hydroxide, achieving high qualitative and quantitative reproducibility for the yeast sample across at least 23 runs. The data underscore the capability of CZE-MS/MS for large-scale quantitative TDP of complex samples, signaling its readiness for deployment in broad biological applications. The MS RAW files were deposited in ProteomeXchange Consortium with the data set identifier of PXD046651.