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

Journal of Separation Science, Journal Year: 2023, Volume and Issue: 46(15)

Published: May 26, 2023

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

Language: Английский

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Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

Electrophoresis, Journal Year: 2023, Volume and Issue: 45(1-2), P. 165 - 198

Published: Sept. 5, 2023

Abstract This review article brings a comprehensive survey of developments and applications high‐performance capillary microchip electromigration methods (zone electrophoresis in free solution or sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, electrochromatography) for analysis, micropreparation, physicochemical characterization peptides the period from 2021 up to ca. middle 2023. Progress study properties various aspects their such as sample preparation, adsorption suppression, electroosmotic flow regulation, detection, are presented. New particular demonstrated, several types reported. They cover qualitative quantitative analysis synthetic isolated determination complex biomatrices, peptide profiling biofluids tissues, monitoring chemical enzymatic reactions changes peptides. include also amino acid sequence peptides, mapping proteins, separation stereoisomers chiral analyses. In addition, micropreparative separations interactions with other (bio)molecules by above CE described.

Language: Английский

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Buffering agents and additives for the background electrolyte solutions used for peptide and protein capillary zone electrophoresis

TrAC Trends in Analytical Chemistry, Journal Year: 2023, Volume and Issue: 164, P. 117080 - 117080

Published: May 6, 2023

The background electrolyte solution (BGE) has a strong impact on the separation performance of peptides and proteins in all techniques governed by electrophoretic principles, due to its considerable influence mechanism physicochemical properties both sample inner surface device, which mostly consists fused-silica capillary tube. This review article examines discusses effects composition pH BGE electrokinetic phenomena taking place at solid liquid interface within capillaries, generating electroosmotic flow (EOF), zone electrophoresis (CZE), is most used mode for these biopolymers. paper reviews literature relevant explain how pH, ionic strength, conductivity employed peptide protein CZE efficiency, selectivity resolution other factors affecting separation, including flow. Also examined discussed compatibility with ionization sources on-line hyphenation mass spectrometry two popular preconcentration methods field-amplified stacking (FASS) dynamic junction.

Language: Английский

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Transient isotachophoresis–Capillary zone electrophoresis–Mass spectrometry method with off-line microscale solid phase extraction pretreatment for quantitation of intact low molecular mass proteins in various biological fluids

Journal of Chromatography A, Journal Year: 2024, Volume and Issue: 1718, P. 464697 - 464697

Published: Feb. 3, 2024

Quantification of proteins is still predominantly done by the traditional bottom-up approach. Targeting intact in complex biological matrices connected with multiple challenges during sample pretreatment, separation, and detection step analytical workflow. In this work, we focused on development an on-line hyphenated capillary zone electrophoresis-mass spectrometry method employing off-line microscale solid-phase extraction based hydrophilic lipophilic balance (HLB) sorbent as a pretreatment for analysis low molecular mass (<20 kDa) spiked various fluids (human serum, plasma, urine, saliva). A detailed optimization process involved selection suitable surface, background electrolyte (BGE), comparison two in-capillary preconcentration methods, namely transient isotachophoresis (tITP) dynamic pH junction (DPJ), to enhance sensitivity method. Optimum separation analytes was achieved using uncoated bare fused silica 500 mM formic acid (pH 1.96) + 5% (v/v) acetonitrile BGE. tITP utilized optimum technique, achieving 19- 127-fold increase signal intensity when 200 ammonium formate (adjusted 4.00) leading BGE terminating electrolyte. Off-line eluate treatment procedures evaluated ensure compatibility selected preconcentration, process. Achieved recoveries were range 76-100% 12-54% 21-106% 25-98% saliva evaporated reconstituted solution achieve The validated across different matrices, offering good linearity, accuracy, precision, making it proteomic studies (e.g., therapeutic drug monitoring, biomarker research) samples.

Language: Английский

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Post‐translational modifications of proteins in cardiovascular diseases examined by proteomic approaches

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

Published: March 5, 2024

Over 400 different types of post‐translational modifications (PTMs) have been reported and over 200 various PTMs discovered using mass spectrometry (MS)‐based proteomics. MS‐based proteomics has proven to be a powerful method capable global PTM mapping with the identification modified proteins/peptides, localization sites quantitation. play regulatory roles in protein functions, activities interactions heart related diseases, such as ischemia/reperfusion injury, cardiomyopathy failure. The recognition that are specific cardiovascular pathology clarification mechanisms underlying these at molecular levels crucial for discovery novel biomarkers application clinical setting. With sensitive MS instrumentation biostatistical methods precise processing data, low‐abundance can successfully detected beneficial or unfavorable effects on cardiac function determined. Moreover, computational proteomic strategies predict based data gained an increasing interest contribute characterization profiles disorders. More recently, machine learning‐ deep learning‐based employed locations explore crosstalk. In this review article, briefly overviewed, approaches identification/quantitation discussed recently published studies associated diseases included.

Language: Английский

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Trends Development and Applications on Electrophoresis Techniques of Slab Gel, Capillary, Microchip/Microfluidic Capillary, and Isotachophoresis

Talanta, Journal Year: 2025, Volume and Issue: unknown, P. 128029 - 128029

Published: April 1, 2025

Language: Английский

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On-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry for the analysis of protein biomarkers in biological fluids and food: A tutorial

Advances in Sample Preparation, Journal Year: 2023, Volume and Issue: 7, P. 100082 - 100082

Published: July 4, 2023

The analysis by capillary electrophoresis (CE) of low abundant proteins in complex samples, such as biological fluids and food, is especially challenging, due to the poor concentration sensitivity microscale separation techniques sample matrix complexity. In order overcome these major drawbacks, microextraction preparation based on on-line solid-phase extraction (SPE-CE) are regarded an excellent alternative for clean-up analyte preconcentration with minimum handling. this study, we present, a tutorial, valve-free aptamer affinity electrophoresis-mass spectrometry (AA-SPE-CE-MS) method purification, preconcentration, separation, detection, characterization intact protein biomarkers food using cases study α‑synuclein (α‑syn), concanavalin A (Con A), β-lactoglobulin (β-LG), which related Parkinson's disease allergy, respectively. This tutorial not limited description analytical method, but it also provides ready-to-use procedures sorbent devices, introduces strategies undesired effects, allowing straightforward implementation optimum performance AA-SPE-CE-MS, platform develop further applications.

Language: Английский

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Greenness of proteomic sample preparation and analysis techniques for biopharmaceuticals

TrAC Trends in Analytical Chemistry, Journal Year: 2023, Volume and Issue: 171, P. 117490 - 117490

Published: Dec. 19, 2023

Proteins are efficient therapeutic agents and attractive potential new biomarkers due to their physiological pathological functions. Further, biopharmaceuticals feature complex protein architectures that must be assayed quality controlled. Capillary electrophoresis (CE) has become one of the most powerful tools for proteomic analysis. CE is an eco-friendlier alternative traditional liquid chromatography; however, its true greenness environmental impact not very often confirmed by green metrics. Here, we assessed bottom-up, middle-up, top-down sample preparation analytical approaches analysis proteins using AGREEprep AGREE metric systems, respectively. The combined use both can help identify weaknesses overall procedure point toward greener alternatives. results showed trends in workflows should focused mainly on miniaturization devices applying in-line on-line preparation.

Language: Английский

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Investigating the Merits of Microfluidic Capillary Zone Electrophoresis–Mass Spectrometry (CZE-MS) in the Bottom-Up Characterization of Larger RNAs

Journal of the American Society for Mass Spectrometry, Journal Year: 2024, Volume and Issue: 35(3), P. 561 - 574

Published: Feb. 13, 2024

Established bottom-up approaches for the characterization of nucleic acids (NAs) rely on strand-cleavage activity nucleotide-specific endonucleases to generate smaller oligonucleotides amenable gas-phase sequencing. The complexity these hydrolytic mixtures calls utilization a front-end separation facilitate full mass spectrometric (MS) characterization. This report explored merits microfluidic capillary zone electrophoresis (CZE) as possible alternative common liquid chromatography techniques. An oligonucleotide ladder was initially employed investigate roles fundamental analyte features and experimental parameters in determining outcome CZE-MS analyses. results demonstrated ability fully resolve various rungs into discrete electrophoretic peaks with full-width half-height (FWHH) resolution that visibly affected by overall amount material injected system. Analogous were obtained from digestion mixture prepared treating yeast tRNAPhe (75 nt) RNase T1, which provided several well-resolved spite increasing sample heterogeneity. regular shapes such peaks, however, belied fact most them contained sets comigrating species, shown corresponding MS spectra. Even though it not segregate each species an individual peak, analysis still proved capable unambiguously identifying total 29 products, sufficient cover 96% tRNAPhe's sequence. Their masses accurately reflected presence modified nucleotides characteristic this type substrate. 364 nt HIV-1 5′-UTR be more challenging. electropherogram displayed fewer significantly greater incidence product comigration. In case, fractionating highly heterogeneous bands helped reduce signal suppression detection bias. As result, data enabled assignment 248 products out 513 predicted sequence, afforded 100% sequence coverage. figure represented significant improvement over 36 identified earlier under suboptimal conditions, only 57% coverage, or 83 observed direct infusion nanospray-MS (72%). These measure excellent potential technique support progressively larger NA samples, putative therapeutics mRNA vaccines.

Language: Английский

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A novel AgNFs/MOF/CFC substrate-based flexible SERS sensor for high-sensitive detection of dye molecules

Engineering Research Express, Journal Year: 2024, Volume and Issue: 6(2), P. 025015 - 025015

Published: June 1, 2024

Abstract Metal–organic framework (MOF) materials have been increasingly used owing to their high porosity, chemical stability, and semiconducting properties. With the help of MOF materials, MOF-based SERS substrates can exhibit sensitivity. Herein, we proposed a flower-like Ag nanoparticles (AgNFs)/metal–organic (MOF)/ carbon fiber cloth (CFC) substrate-based surface-enhanced Raman scattering (SERS) sensor for high-sensitive detection dye molecules. is as signal amplifier accelerate approach molecules near AgNFs on surface anchor substrate surface. The occurred AgNFs/MOF/CFC substrate, providing an enhancement factor (EF) 5.53 × 10 7 . Benefitting from amplification function superiority SERS, this represented sensitivity performance wide range. Importantly, combination Co elements with silver in composite enhances resistance oxidation by atmospheric moisture resulting excellent stability (the maintained good activity after 45 days exposure air) substrate. basic was evaluated using rhodamine 6 G (R6G). (Detection limit reached −12 M) TC 0.1 ppm. This method provides potential cost-effective strategy develop advanced sensing devices effectively monitor pollutants environmental waters.

Language: Английский

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