Capillary Electrophoresis-mass Spectrometry Research Articles

#2561 URINE PROTEOMICS FOR PREDICTION OF DISEASE PROGRESSION IN PATIENTS WITH IGA NEPHROPATHY

Abstract Background and Aims IgA nephropathy (IgAN) may lead to kidney failure. The urinary proteomics biomarker-based classifier (IgAN237) may predict progression at the time of kidney biopsy [1]. We now studied whether IgAN237 predicts disease progression not only at the time of biopsy but also later in the course of IgAN. Method Urine samples from 103 patients with biopsy-proven IgAN were analyzed using capillary electrophoresis-mass spectrometry at baseline (IgAN237 score-1) and 89 also at follow-up (IgAN237 score-2). Patients were grouped into ‘non-progressors’ (IgAN237 score ≤0.38 Units) and ‘progressors’ (IgAN237 score >0.38 Units). Historical and follow-up data included, e.g., estimated glomerular filtration rate (eGFR)-slopes, urinary albumin/creatinine ratio (UACR)-slopes and medication. Multiple logistic regression analysis, using a stepwise model, was done with progressors and non-progressors as dependent factors including explanatory variables age, sex, eGFR- and UACR-slopes. Results Median age at biopsy was 44 years (range 11-92); 63% were male. Median interval between biopsy and IgAN237 score-1 was 65 months (0-606), and between IgAN237 score-1 and score-2 was 258 days (71-531). IgAN237 score-1 and score-2 values did not differ significantly and were correlated (rho = 0.44, p<0.001). Twenty-eight and 26% of patients were classified as progressors based on IgAN237 score-1 and score-2, respectively. The IgAN237 score inversely correlated with the chronic eGFR slopes (rho = -0.278, p = 0.02 for score-1; rho = -0.409, p = 0.002 for score-2) and with ±180days eGFR slopes (rho = -0.31, p = 0.009 and rho = -0.439, p = 0.001, respectively) (Figure 1). The ±180days eGFR-slopes were more reduced for progressors than non-progressors (median -5.98 versus -1.22 mL/min/1.73m2 per year for score-1, p<0.001; -3.02 vs 1.08 mL/min/1.73m2 per year for score-2, p = 0.047) (Figure 2). The only significant variable maintained in a multiple logistic regression analysis for IgAN237 score-1 (progressor vs non-progressor) was ±180days eGFR slope (p<0.001) and for IgAN237 score-2 the UACR (p = 0.002) and age at baseline (p = 0.016). Conclusion The urinary IgAN237 classifier represents a risk stratification tool in IgAN not only at the time of biopsy but also later in the course of the disease. It may guide physicians in management and follow-up strategies in an individualized manner.

Capillary electrophoresis-mass spectrometry for protein analyses under native conditions: Current progress and perspectives.

Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the protein higher order structure. The coupling with electromigration separation techniques under native conditions enables the characterization of proteoforms and highly complex protein mixtures. In this review, we present an overview of current native CE-MS technology. First, the status of native separation conditions is described for capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE), and capillary isoelectric focusing (CIEF), as well as their chip-based formats, including essential parameters such as electrolyte composition and capillary coatings. Further, conditions required for native ESI-MS of (large) protein constructs, including instrumental parameters of QTOF and Orbitrap systems, as well as requirements for native CE-MS interfacing are presented. On this basis, methods and applications of the different modes of native CE-MS are summarized and discussed in the context of biological, medical, and biopharmaceutical questions. Finally, key achievements are highlighted and concluded, while remaining challenges are pointed out.

Exploring proteoforms of the IgG2 monoclonal antibody panitumumab using microchip capillary electrophoresis-mass spectrometry

The IgG2 type monoclonal antibody panitumumab is an anti-epidermal growth factor receptor (EGFR) drug used for the treatment of EGFR-expressing, chemotherapy resistant, metastatic colorectal carcinoma. In this study, panitumumab drug product was first analysed using size exclusion chromatography coupled to mass spectrometry for rapid identity testing. The experimental data led to the identification of two panitumumab isoforms with several prominent forms remaining unidentified, despite apparently low sample complexity. Microchip capillary electrophoresis-mass spectrometry (CE-MS) was subsequently utilised for a more detailed characterization. It was observed that panitumumab is subject to partial N-terminal pyroglutamate formation. Incomplete conversion is uncharacteristic for N-terminally exposed glutamines and in case of panitumumab gives rise to forms which show successive mass offsets of 17 Da, respectively. If not separated before mass spectrometric analysis, e.g. by capillary electrophoresis, such near isobaric species coalesce into single MS peaks, which subsequently hampers or prevents their assignment. With a total of 42 panitumumab isoforms assigned by CE-MS, these observations highlight a potential pitfall of commonly applied rapid identity testing workflows and demonstrate that even low complexity biopharmaceuticals can require separation strategies which offer high separation selectivity for species close in mass.

Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

The Fatty Liver, Cirrhosis, and Liver Cancer Study (TENDENCY): Protocol for a Multicenter Case-Control Study.

Hepatocellular cancer (HCC) is associated with high mortality, and early diagnosis leads to better survival. Patients with cirrhosis, especially due to nonalcoholic fatty liver disease and viral hepatitis, are at higher risk of developing HCC and form the main screening group. The current screening methods for HCC (6-monthly screening with serum alpha fetoprotein and ultrasound liver) have low sensitivity; hence, there is a need for better screening markers for HCC. Our study, TENDENCY, aims to validate the novel screening markers (methylated septin 9, urinary volatile organic compounds, and urinary peptides) for HCC diagnosis and study these noninvasive biomarkers in liver disease. This is a multicenter, nested case-control study, which involves comparing the plasma levels of methylated septin 9 between confirmed HCC cases and patients with cirrhosis (control group). It also includes the comparison of urine samples for the detection of HCC-specific volatile organic compounds and peptides. Based on the findings of a pilot study carried out at University Hospital Coventry & Warwickshire, we estimated our sample size to be 308 (n=88, 29% patients with HCC; n=220, 71% patients with cirrhosis). Urine and plasma samples will be collected from all participants and will be frozen at -80 °C until the end of recruitment. Gas chromatography-mass spectrometry will be used for urinary volatile organic compounds detection, and capillary electrophoresis-mass spectrometry will be used for urinary peptide identification. Real-time polymerase chain reaction will be used for the qualitative detection of plasma methylated septin 9. The study will be monitored by the Research and Development department at University Hospital Coventry & Warwickshire. The recruitment stage was completed in March 2023. The TENDENCY study is currently in the analysis stage, which is expected to finish by November 2023. There is lack of effective screening tests for hepatocellular cancer despite higher mortality rates. The application of more sensitive plasma and urinary biomarkers for hepatocellular cancer screening in clinical practice will allow us to detect the disease at earlier stages and hence, overall, improve HCC outcomes. DERR1-10.2196/44264.

Capillary electrophoresis-mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018-March 2023).

Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis-mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in-depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis-mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis-mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018-March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.

Production and characterization of a camelid single domain anti-CD22 antibody conjugated to DM1.

Antibody drug conjugates (ADCs) with twelve FDA approved drugs, known as a novel category of anti-neoplastic treatment created to merge the monoclonal antibody specificity with cytotoxicity effect of chemotherapy. However, despite many undeniable advantages, ADCs face certain problems, including insufficient internalization after binding, complex structures and large size of full antibodies especially in targeting of solid tumors. Camelid single domain antibody fragments (Nanobody®) offer solutions to this challenge by providing nanoscale size, high solubility and excellent stability, recombinant expression in bacteria, in vivo enhanced tissue penetration, and conjugation advantages. Here, an anti-human CD22 Nanobody was expressed in E.coli cells and conjugated to Mertansine (DM1) as a cytotoxic payload. The anti-CD22 Nanobody was expressed and purified by Ni-NTA resin. DM1 conjugated anti-CD22 Nanobody was generated by conjugation of SMCC-DM1 to Nanobody lysine groups. The conjugates were characterized using SDS-PAGE and Capillary electrophoresis (CE-SDS), RP-HPLC, and MALDI-TOF mass spectrometry. Additionally, flow cytometry analysis and a competition ELISA were carried out for binding evaluation. Finally, cytotoxicity of conjugates on Raji and Jurkat cell lines was assessed. The drug-to-antibody ratio (DAR) of conjugates was calculated 2.04 using UV spectrometry. SDS-PAGE, CE-SDS, HPLC, and mass spectrometry confirmed conjugation of DM1 to the Nanobody. The obtained results showed the anti-CD22 Nanobody cytotoxicity was enhanced almost 80% by conjugation with DM1. The binding of conjugates was similar to the non-conjugated anti-CD22 Nanobody in flow cytometry experiments. Concludingly, this study successfully suggest that the DM1 conjugated anti-CD22 Nanobody can be used as a novel tumor specific drug delivery system.

Annual review of capillary electrophoresis technology in 2022

This article provides a detailed review of capillary electrophoresis (CE) technology in 2022, summarizing a total of 881 CE technology-related articles searched from ISI Web of Science using the keywords "capillary electrophoresis mass spectrometry" or "capillary isoelectric focusing" or "micellar electrokinetic chromatography" or "capillary electrophoresis" (excluding "capillary electrochromatography""microchip" "microfluidic" "capillary monolithic column"). The review focuses on 16 articles published in Lancet Global Health, ACS Central Science, Microbiome, Trends in Food Science & Technology, TrAC-Trends in Analytical Chemistry, Journal of Pharmaceutical Analysis, Journal of Cachexia, Sarcopenia and Muscle, Food Hydrocolloids, Science of the Total Environment, and Carbohydrate Polymers with impact factors (IFs) greater than 10.0, and 46 articles published in Analytical Chemistry, Analytica Chimica Acta, Talanta, and Food Chemistry with IFs between 5.0 and 10.0. A comprehensive overview of representative CE works published in Journal of Chromatography A, Electrophoresis, and important Chinese core journals (Peking University) with IFs<5.0 is also provided. Based on IFs, this review introduces representative works on CE to facilitate readers' understanding of important research advances in CE technology over the last year.

Pro-tolerogenic gut microbiota induce rapid and distinct gut metabolic changes after cardiac allografting

Abstract Desulfovibrio desulfuricans (Desulfo) induces pro-inflammatory effects, while Bifidobacterium pseudolongum (Bifido) induces pro-tolerant effects. Bifido results in decreased cardiac allograft inflammation and fibrosis compared to Desulfo. Lymph node (LN) laminins α4 and α5 are differentially regulated in transplant tolerance (α4&amp;gt;α5) and immunity (α4&amp;lt;α5). We investigated whether specific microbiota alter LN architecture, immune cell content, and gut metabolites. B6 mice received antibiotics followed by bacteria gavage (Bifido, Desulfo, or PBS control), BALB/c heart transplants, and daily tacrolimus. LNs were assessed by immunohistochemistry. Stool metabolome profiling used capillary electrophoresis-mass spectrometry with partial least square-discriminant analyses. Desulfodecreased LN laminin α4:α5 ratios and Foxp3+ regulatory T cells (Tregs) after 7 days. After 14 days, Desulfotreated animals had greater allograft fibrosis while Bifidoincreased LN laminin α4:α5 ratios and Tregs. Gut metabolomes demonstrated highly distinct clustering between groups after 7 days. Bifidotreatment enriched compounds related to polyamine metabolism (spermine, spermidine, and putrescine), as well as by pathway analysis (arginine and proline plus spermidine and spermine metabolism) compared to Desulfoand control. Overall, Desulfoinduced rapid pro-inflammatory changes (decreased LN laminin ratios and Tregs, increased graft fibrosis), while Bifidoinduced rapid pro-tolerant changes (increased LN laminin ratios and Tregs). Bifidomodulated distinct polyamine pathway metabolites, compounds critical for T cell reprogramming. These results suggest polyamines as mediators of Bifidopro-tolerogenic effects. This work is supported by grants from NIH (R01HL148672, U01AI170050, and T32AI95190-10).

Accurate determination of the milk protein allergen β-lactoglobulin by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method was developed to purify, preconcentrate, separate, and characterize the milk allergenic protein β-lactoglobulin (β-LG) in food samples. The sorbent to pack into the SPE microcartidges was prepared by immobilizing an aptamer against β-LG onto magnetic bead particles. After optimizing the SPE-CE-MS method, the sample (ca. 75 μL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while a solution of 100 mM NH4OH (pH 11.2) (ca. 100 nL) was used for the protein elution. The linearity of the method ranged between 0.1 and 20 μg mL−1 and the limit of detection (LOD) was 0.05 μg mL−1, which was 200 times lower than by CE-MS. The method was repeatable in terms of relative standard deviation (RSD) for migration times and peak areas (<0.5% and 2.4%, respectively) and microcartridge lifetime was more than 25 analyses. The applicability of the method for the determination of low levels of β-LG was shown by analyzing milk-free foods (i.e. a 100% cocoa dark chocolate, a hypoallergenic formula for infants, and a dairy-free white bread) and milk-containing white breads. Results were satisfactory in all cases, thus demonstrating the great potential of the developed method for accurate food safety and quality control.

Determination of a lectin protein allergen in food by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An aptamer affinity sorbent was prepared for clean-up, preconcentration, separation and characterization of a food allergen protein by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). SPE microcartridges were packed with a sorbent based on magnetic bead particles modified with an aptamer against the target lectin protein concanavalin A (Con A). After optimization of several parameters of the SPE-CE-MS method, the sample (ca. 30 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while the retained protein was eluted with 100 mM NH4OH (pH 11.2) (ca. 100 nL). The developed method was linear between 0.5 and 20 mg·L-1 and the limit of detection (LOD) was 0.25 mg·L-1, which was 100 times lower than by CE-MS. The repeatability of the method was satisfactory, with relative standard deviations (RSD) for migration times and peak areas below 1.9 and 8.1%, respectively. In addition, the microcartridges could be reused more than 25 analyses without significant loss of extraction efficiency. Finally, the applicability and versatility of the developed method were demonstrated by analyzing low levels of Con A in different food matrices (i.e. white beans, as well as chickpea, lentils, and wheat flours), leading to satisfactory results, with recoveries between 87 and 115%.

5613004 NEWBORN SCREENING FOR SICKLE CELL DISEASE IN GERMANY - THE FIRST YEAR

Newborn screening is the first and most important step in secondary prevention in children with sickle cell disease. Serious complications and deaths can be almost completely prevented by ensuring the necessary preventive measures against infection (vaccinations and penicillin prophylaxis) and with the help of training measures that enable parents to recognize the symptoms of acute anemia/hemolysis in good time. In 2017, as part of a consensus conference with over 50 participants from 13 nations, the requirements that a newborn screening program for sickle cell disease should meet were defined. In Germany, we have consistently implemented these requirements. In addition, a total of five pilot studies involving almost 220,000 participants were carried out. These studies have shown that if sickle cell disease were part of newborn screening, it would probably be one of the most prevalent target diseases. On the basis of the consensus document and the epidemiological data collected for the first time, the relevant institutions and authorities could ultimately be convinced of the usefulness of the screening. The amendment to the law required for the introduction of screening came into force on September 30, 2021. Since then, all children born in Germany have also been screened for sickle cell disease as part of the national newborn screening program. Due to the federal organization of the German state, there is still no official evaluation of the screening results. However, we conducted a survey among the eleven German screening laboratories and the 47 pediatric haematology centers that will take on the long-term care of the sick children. The data indicate that in the first year, approximately 120-150 of 800,000 newborns have sickle cell disease. The German authorities have approved HPLC, capillary electrophoresis and tandem mass spectrometry as measurement methods in screening. Deviating from this, some laboratories use molecular genetic methods to screen for the HbS mutation. In the laboratories participating in our survey, 13 false-positive findings were recorded during the survey period. 4 children had thalassemia major, which is not formally a target disease but is of course definitely worth reporting. Four children were HbS heterozygous. Three children had very rare hemoglobin variants and in three children we have no feedback on the false positive finding. We assume that the heterozygotes and the rare variants were reported out of uncertainty and fall under the “starting difficulties” category. These false positives all occurred within the first few weeks of the program and could be eliminated by retraining the laboratory staff. Conclusion: Not only, but also thanks to the constant support of our European colleagues, especially in the form of the 2017 consensus paper, we have succeeded in introducing newborn screening for sickle cell disease. The first preliminary data show that sickle cell disease, with a prevalence of 1:5000 - 1:6000, will be one of the top diagnoses in screening even in Germany.

On-line aptamer affinity solid-phase extraction direct mass spectrometry for the rapid analysis of α-synuclein in blood

On-line aptamer affinity solid-phase extraction direct mass spectrometry (AA-SPE-MS) is presented for the rapid purification, preconcentration, and characterization of α-synuclein (α-syn), which is a protein biomarker related to Parkinson's disease. Valve-free AA-SPE-MS is easily implemented using the typical SPE microcartridges and instrumental set-up necessary for on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). The essential requirement is substituting the application of the separation voltage by a pressure of 100 mbar for mobilization of the eluted protein through the capillary towards the mass spectrometer. Under optimized conditions with recombinant α-syn, repeatability is good in terms of migration time and peak area (percent relative standard deviation (%RSD) values (n = 3) are 1.3 and 6.6% at 1 μg mL−1, respectively). The method is satisfactorily linear between 0.025 and 5 μg mL−1 (R2 > 0.986), and limit of detection (LOD) is 0.02 μg mL−1 (i.e. 1000, 500, and 10 times lower than by CE-MS, direct MS, and AA-SPE-CE-MS, respectively). The established AA-SPE-MS method is further compared with AA-SPE-CE-MS, including for the analysis of α-syn in blood. The comparison discloses the advantages and disadvantages of AA-SPE-MS for the rapid and sensitive targeted analysis of protein biomarkers in biological fluids.

Highly-sensitive label-free deep profiling of N-glycans released from biomedically-relevant samples

Alterations of protein glycosylation can serve as sensitive and specific disease biomarkers. Labeling procedures for improved separation and detectability of oligosaccharides have several drawbacks, including incomplete derivatization, side-products, noticeable desialylation/defucosylation, sample loss, and interference with downstream analyses. Here, we develop a label-free workflow based on high sensitivity capillary zone electrophoresis-mass spectrometry (CZE-MS) for profiling of native underivatized released N-glycans. Our workflow provides a >45-fold increase in signal intensity compared to the conventional CZE-MS approaches used for N-glycan analysis. Qualitative and quantitative N-glycan profiling of purified human serum IgG, bovine serum fetuin, bovine pancreas ribonuclease B, blood-derived extracellular vesicle isolates, and total plasma results in the detection of >250, >400, >150, >310, and >520 N-glycans, respectively, using injected amounts equivalent to <25 ng of model protein and nL-levels of plasma-derived samples. Compared to reported results for biological samples of similar amounts and complexity, the number of identified N-glycans is increased up to ~15-fold, enabling highly sensitive analysis of sample amounts as low as sub-0.2 nL of plasma volume equivalents. Furthermore, highly sialylated N-glycans are identified and structurally characterized, and untreated sialic acid-linkage isomers are resolved in a single CZE-MS analysis.

Electrochemically Etched Tapered-Tip Stainless-Steel Electrospray-Ionization Emitters for Capillary Electrophoresis-Mass Spectrometry.

We have used household consumables to facilitate electrochemical etching of stainless-steel hypodermic tubing to produce tapered-tip emitters suitable for electrospray ionization for use in mass spectrometry. The process involves the use of 1% oxalic acid and a 5 W USB power adapter, commonly known as a phone charger. Further, our method avoids the otherwise commonly used strong acids that entail chemical hazards: concentrated HNO3 for etching stainless steel, or concentrated HF for etching fused silica. Hence, we here provide a convenient and self-inhibiting procedure with minimal chemical hazards to manufacture tapered-tip stainless-steel emitters. We show its performance in metabolomic analysis with CE-MS of a tissue homogenate where the metabolites acetylcarnitine, arginine, carnitine, creatine, homocarnosine, and valerylcarnitine were identified, all with basepeak separated electropherograms, within <6 min of separation. The mass spectrometry data are freely available through the MetaboLight public data repository via access number MTBLS7230.

Effect of Exogenous Acute β-Hydroxybutyrate Administration on Different Modalities of Exercise Performance in Healthy Rats.

A ketone body (β-hydroxybutyrate [β-HB]) is used as an energy source in the peripheral tissues. However, the effects of acute β-HB supplementation on different modalities of exercise performance remain unclear. This study aimed to assess the effects of acute β-HB administration on the exercise performance of rats. In study 1, Sprague-Dawley rats were randomly divided into six groups: endurance exercise (EE + PL and EE + KE), resistance exercise (RE + PL and RE + KE), and high-intensity intermittent exercise (HIIE + PL and HIIE + KE) with placebo (PL) or β-HB salt (KE) administration. In study 2, metabolome analysis using capillary electrophoresis mass spectrometry was performed to profile the effects of β-HB salt administration on HIIE-induced metabolic responses in the skeletal and heart muscles. The maximal carrying capacity (rest for 3 min after each ladder climb, while carrying heavy weights until the rats could not climb) in the RE + KE group was higher than that in the RE + PL group. The maximum number of HIIE sessions (a 20-s swimming session with a 10-s rest between sessions, while bearing a weight equivalent to 16% of body weight) in the HIIE + KE group was higher than that in the HIIE + PL group. However, there was no significant difference in the time to exhaustion at 30 m·min -1 between the EE + PL and the EE + KE groups. Metabolome analysis showed that the overall tricarboxylic acid cycle and creatine phosphate levels in the skeletal muscle were higher in the HIIE + KE group than those in the HIIE + PL group. These results indicate that acute β-HB salt administration may accelerate HIIE and RE performance, and the changes in metabolic responses in the skeletal muscle after β-HB salt administration may be involved in the enhancement of HIIE performance.

Two-Dimensional Capillary Zone Electrophoresis-Mass Spectrometry: Intact mAb Charge Variant Separation Followed by Peptide Level Analysis Using In-Capillary Digestion.

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.

Sensitive Analysis of Recombinant Human Erythropoietin Glycopeptides by On-Line Phenylboronic Acid Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry.

In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O126 and N83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO2-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO2-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N24 and N38 glycopeptides. Finally, the established method was used to analyze two rhEPO products (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CE-MS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of the protein digest.

Coupling capillary electrophoresis-electrospray ionization mass spectrometry with sheathless interface for drug analysis

Capillary electrophoresis-mass spectrometry （CE-MS） combines the advantages of capillary electrophoresis， such as the high separation efficiency and low sample consumption， and the high detection sensitivity of mass spectrometry and the ability for providing the structural information for structure elucidation of unknown components. However， the interface technology for coupling capillary electrophoresis and mass spectrometry is still not well resolved. In the present work， we explored the application of the sheathless CE-MS interface which was prepared by gold foil-wrapped CE separation column tip directly as a spray electrode for the analysis of five tyrosine kinase inhibitors， namely sunitinib， imatinib mesylate， gefitinib， dasatinib and erlotinib. This interface integrates separation and electrospray ionization in one capillary， which is easy to manufacture， low in cost， and can be produced in batches. We found that using the nonaqueous CE separation mode can not only achieve baseline separation of five tyrosine kinase inhibitors， but also obtain stable mass spectrometry signals. First， we investigated the effect of the electrolyte solution composition on the separation. The optimized background electrolyte composition was obtained： 2% （v/v） acetic acid and 5 mmol/L ammonium acetate in acetonitrile-methanol （80∶20， v/v）. Under optimized conditions， the five kinase inhibitors could be baseline separated， meantime， the sheathless interface could also maintain stable electrospray for a long time. The relative standard deviation （RSD） values of the intraday and interday repeatability of the analyte retention times were less than 0.5% and 0.8%， respectively， and the RSD value between interface batches is less than 2.6%. Compared with CE-MS with aqueous phase， the separation column efficiency of the five tyrosine kinase inhibitors under nonaqueous phase conditions is higher， the detection sensitivity is higher， and the absolute detection limit reaches amol level. In addition， we evaluated the sheathless interface with various organic acids， such as palaflin A， salvianolic acid C， and rosmarinic acid， as well as hydrophobic macrolide antibiotics， azithromycin， erythromycin， and sporin A， good separation effect and mass spectrometric detection results can be obtained.

Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues.

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5 and 2-PP-InsP5. The latter has not previously been described in nature. The analysis of feces and the commercial mouse diet suggests that the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5 and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.