Imaged capillary isoelectric focusing and online mass spectrometry for milk whey protein characterization in dairy products

Analytical Biochemistry, Journal Year: 2025, Volume and Issue: 699, P. 115765 - 115765

Published: Jan. 6, 2025

Language: Английский

---

An integrated protocol based on workflows of imaged capillary isoelectric focusing (icIEF) for in-depth protein heterogenous characterization: high-efficient fractionation and online mass spectrometry detection

Green Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 100204 - 100204

Published: Jan. 1, 2025

Language: Английский

---

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Analytical Chemistry, Journal Year: 2022, Volume and Issue: 94(27), P. 9674 - 9682

Published: June 29, 2022

Protein complexes are the functional machines in cell and heterogeneous due to protein sequence variations post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering microheterogeneity of intact complexes. The method exhibited superior separation resolution than conventional native zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations MS characterization seven different forms a streptavidin homotetramer with N-terminal methionine removal, acetylation, formylation four carbonic anhydrase-zinc complex arising from PTMs (succinimide, deamidation, etc.). addition, resolved states interchain cysteine-linked antibody-drug conjugate (ADC1) as new class anticancer therapeutic agents that bears distribution varied drug-to-antibody ratio (DAR) species. More importantly, enabled precise measurements points (pIs) complexes, which reflect surface electrostatic properties We studied how variations/PTMs modulate pIs drug loading affects antibodies. discovered keeping residue one subunit decreased its pI by 0.1, adding acetyl group onto reduced nearly 0.4, incorporating formyl around 0.3, two more molecules on ADC1 molecule increased 0.1. data render valuable tool delineating

Language: Английский

---

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

PROTEOMICS, Journal Year: 2023, Volume and Issue: 24(3-4)

Published: June 13, 2023

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

Language: Английский

---

Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity

Analytica Chimica Acta, Journal Year: 2024, Volume and Issue: 1297, P. 342349 - 342349

Published: Feb. 12, 2024

Language: Английский

---

Mass Spectrometry-Based Charge Heterogeneity Characterization of Therapeutic mAbs with Imaged Capillary Isoelectric Focusing and Ion-Exchange Chromatography as Separation Techniques

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(4), P. 2548 - 2560

Published: Jan. 19, 2023

Imaged capillary isoelectric focusing (icIEF) and ion-exchange chromatography (IEX) are two essential techniques that routinely used for charge variant analysis of therapeutic monoclonal antibodies (mAbs) during their development in quality control. These separate mAb variants based on different mechanisms IEX have been developed as front-end separation online mass spectrometry (MS) detection, which is robust intact protein identification. Recently, an innovative, coupled icIEF-MS technology has constructed our laboratory. In this study, strong cation exchange (SCX)-MS were optimized heterogeneity characterization a diverse mAbs results compared methodological validation. It was found outperformed SCX-MS study by demonstrating outstanding sensitivity, low carryover effect, accurate identification, higher resolution although contributed to throughput. Ultimately, integrating novel icIEF-HRMS with the more common can provide promising comprehensive strategy accelerating complex therapeutics.

Language: Английский

---

Coupling High-Field Asymmetric Waveform Ion Mobility Spectrometry with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(25), P. 9497 - 9504

Published: May 31, 2023

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has emerged as an essential technique for top-down proteomics (TDP), providing superior separation efficiency and high detection sensitivity proteoform analysis. Here, we aimed to further enhance the performance of CZE-MS/MS TDP via coupling online gas-phase fractionation using high-field asymmetric waveform ion mobility (FAIMS). When compensation voltage (CV) FAIMS was changed from −50 30 V, median identified proteoforms increased less than 10 kDa about kDa, suggesting that can efficiently fractionate by their size. CZE-FAIMS-MS/MS boosted number identifications a yeast sample nearly 3-fold relative alone. It particularly benefited identification relatively large proteoforms, improving in range 20–45 6-fold compared gained 20-fold better signal-to-noise ratios randomly selected no FAIMS. We expect will be useful tool advancing coverage TDP. This work shows first example CE with (IMS)

Language: Английский

---

Multidimensional separations in top–down proteomics

Analytical Science Advances, Journal Year: 2023, Volume and Issue: 4(5-6), P. 181 - 203

Published: May 29, 2023

Abstract Top–down proteomics (TDP) identifies, quantifies, and characterises proteins at the intact proteoform level in complex biological samples to understand function cellular mechanisms. However, analysing using TDP is still challenging due high sample complexity wide dynamic range. High‐resolution separation methods are often applied prior mass spectrometry (MS) analysis decrease increase throughput. These methods, however, may not be efficient enough characterise low abundance proteoforms samples. As such, multidimensional techniques (combination of two or more with orthogonality) have been developed that demonstrate improved resolution comprehensive identification TDP. A suite couple various types liquid chromatography (LC), capillary electrophoresis (CE), and/or gel electrophoresis‐based approaches analyse Here, we reviewed strategies employed for TDP, summarised current applications, discussed gaps addressed future.

Language: Английский

---

Mass spectrometry-intensive top-down proteomics: an update on technology advancements and biomedical applications

Analytical Methods, Journal Year: 2024, Volume and Issue: 16(28), P. 4664 - 4682

Published: Jan. 1, 2024

Substantial progress in the technological development of mass spectrometry-based top-down proteomics enabled broad biomedical applications for bettering our understanding proteoform function modulating diseases and development.

Language: Английский

---

Imaged Capillary Isoelectric Focusing Coupled to High‐Resolution Mass Spectrometry (icIEF‐MS) for Cysteine‐Linked Antibody–Drug Conjugate (ADC) Heterogeneity Characterization Under Native Condition

Electrophoresis, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 30, 2024

ABSTRACT Native mass spectrometry (nMS) is a cutting‐edge technique that leverages electrospray ionization MS (ESI‐MS) to investigate large biomolecules and their complexes in solution. The goal of nMS retain the native structural features interactions analytes during transition gas phase, providing insights into natural conformations. In biopharmaceutical development, serves as powerful tool for analyzing complex protein heterogeneity, allowing examination non‐covalently bonded assemblies state closely resembles folded form. Herein, we present an imaged capillary isoelectric focusing–MS (icIEF–MS) workflow characterize cysteine‐linked antibody–drug conjugate (ADC) under conditions. Two ADCs were analyzed: latest generation ADC polatuzumab vedotin first FDA‐approved brentuximab vedotin. This benefits from recently developed icIEF system MS‐friendly capable directly coupling high‐sensitivity instrument. Results show separation influenced by both drug payloads post‐translational modifications (PTMs), which are then promptly identified MS. Overall, this icIEF–MS method demonstrates potential understand control critical quality attributes (CQAs) essential safe effective use ADCs.

Language: Английский

---