Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip‐based capillary electrophoresis–mass spectrometry

Deyanova, Ekaterina G.; Huang, Richard Y.‐C.; Madia, Priyanka A.; Nandi, Pradyot; Gudmundsson, Olafur; Chen, Guodong

doi:10.1002/elps.202000132

Cited by 10 publications

(6 citation statements)

References 19 publications

(20 reference statements)

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“…Microscale electrokinetics (EK) methods offer great potential for the manipulation and analysis of target bioparticlesincluding not only macromolecules but also nanoparticles and microparticles. These methods combine the attractive features of microfluidics (e.g., portability, low cost, and short response time) with the robustness and simplicity of EK phenomena to achieve label-free particle manipulation as function of the electric properties of particles and suspending solution. − EK-driven microfluidics have been successfully applied for the detection of DNA and proteins, − enrichment and assessment of cancer cells, − electrofusion of mammalian cells to create hybrid cells, and separation of microparticles and cells. − …”

Section: Introductionmentioning

confidence: 99%

Fine-Tuning the Characteristic of the Applied Potential To Improve AC-iEK Separations of Microparticles

Ahamed,

Mendiola-Escobedo,

Ernst

et al. 2023

Anal. Chem.

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There is an immediate need for the development of rapid and reliable methods for microparticle and cell assessments, and electrokinetic (EK) phenomena can be exploited to meet that need in a low cost and label-free fashion. The present study combines modeling and experimentation to separate a binary mixture of microparticles of the same size (5.1 μm), shape (spherical), and substrate material (polystyrene), but with a difference in particle zeta potentials of only ∼14 mV, by applying direct current (DC)-biased low-frequency alternating current (AC) voltages in an insulator-based-EK (iEK) system. Four distinct separations were carried out to systematically study the effect of fine-tuning each of the three main characteristics of the applied voltage: frequency, amplitude, and DC bias. The results indicate that fine-tuning each parameter improved the separation from an initial separation resolution R s = 0.5 to a final resolution R s = 3.1 of the fully fine-tuned separation. The separation method exhibited fair reproducibility in retention time with variations ranging from 6 to 26% between experimental repetitions. The present study demonstrates the potential to extend the limits of iEK systems coupled with carefully fine-tuned DC-biased low-frequency AC voltages to perform discriminatory micron-sized particle separations.

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Section: Introductionmentioning

confidence: 99%

Fine-Tuning the Characteristic of the Applied Potential To Improve AC-iEK Separations of Microparticles

Ahamed,

Mendiola-Escobedo,

Ernst

et al. 2023

Anal. Chem.

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“…A similar ZipChip mCZE‐MS approach was implemented by Deyanova et al. [241] for monitoring the glycosylation profile of a fusion protein with complex sialylated glycoprofile within 6 min. The method can be used to estimate the relative abundance of each glycoform, generating signature fingerprints for the molecule.…”

Section: Applicationsmentioning

confidence: 99%

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

Maráková

Opetová

Tomašovský

2023

J of Separation Science

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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.

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“…The platform achieved baseline separations of eight different charge variants and detected over 200 cetuximab proteoforms within 14 min. Deyanova et al (2021) also developed a CZE‐MS method based on the ZipChip technique for fast and efficient characterization of a highly glycosylated fusion protein, resulting in baseline separations of 12 peaks corresponding to different proteoforms in 6 min (Figure 9). These studies demonstrate the power of CZE‐MS for high‐throughput and comprehensive characterization of therapeutic proteins.…”

Section: Ce‐ms For Multilevel Proteomics Applicationsmentioning

confidence: 99%

“…CE‐MS electropherograms of a fusion protein analyzed by the ZipChip technique with commercially available separation buffer (A), and the optimized separation buffer containing 10% 2‐propanol and 0.2% acetic acid (pH 3.2) (B). Reproduced from Deyanova et al (2021) with permission from John Wiley and Sons, copyright (2021). CE‐MS, capillary electrophoresis‐mass spectrometry [Color figure can be viewed at wileyonlinelibrary.com]…”

Section: Ce‐ms For Multilevel Proteomics Applicationsmentioning

confidence: 99%

Recent advances (2019–2021) of capillary electrophoresis‐mass spectrometry for multilevel proteomics

Chen

McCool

Yang

et al. 2021

Mass Spectrometry Reviews

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Multilevel proteomics aims to delineate proteins at the peptide (bottom-up proteomics), proteoform (top-down proteomics), and protein complex (native proteomics) levels.Capillary electrophoresis-mass spectrometry (CE-MS) can achieve highly efficient separation and highly sensitive detection of complex mixtures of peptides, proteoforms, and even protein complexes because of its substantial technical progress. CE-MS has become a valuable alternative to the routinely used liquid chromatography-mass spectrometry for multilevel proteomics. This review summarizes the most recent (2019-2021) advances of CE-MS for multilevel proteomics regarding technological progress and biological applications. We also provide brief perspectives on CE-MS for multilevel proteomics at the end, highlighting some future directions and potential challenges.capillary isoelectric focusing-mass spectrometry, capillary zone electrophoresis-mass spectrometry, multilevel proteomics, protein complex, proteoform | INTRODUCTIONProteomics aims to characterize proteins in cells comprehensively as a function of biological conditions, including but not limited to their expression, posttranslational modifications (PTMs), interactions, locations, and turnover (Aebersold & Mann, 2016;Zhang et al., 2013). Mass spectrometry (MS)-based proteomics has become crucial for pursuing better understandings of molecular mechanisms of fundamental cellular processes and disease development (Aebersold & Mann, 2016). There are three main strategies: bottom-up proteomics (BUP), top-down proteomics (TDP), and native proteomics.BUP is the most mature and widely used strategy. It identifies proteins through the MS and tandem MS (MS/MS) measurements of peptides derived from the enzymatic

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Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip‐based capillary electrophoresis–mass spectrometry

Cited by 10 publications

References 19 publications

Fine-Tuning the Characteristic of the Applied Potential To Improve AC-iEK Separations of Microparticles

Fine-Tuning the Characteristic of the Applied Potential To Improve AC-iEK Separations of Microparticles

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

Recent advances (2019–2021) of capillary electrophoresis‐mass spectrometry for multilevel proteomics

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