Chromophoreasy, an Excel-Based Program for Detection and Integration of Peaks from Chromatographic and Electromigration Techniques

Vaz, Fernando Antônio Simas; Neves, Leandra N. O.; Marques, Rafael; Sato, Renata Takabayashi; Oliveira, Marcone Augusto Leal de

doi:10.5935/0103-5053.20160076

Cited by 9 publications

(9 citation statements)

References 27 publications

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“… a Error is represented by the standard deviation. b Comparison of peak tailing (i.e., tailing factor at 5% peak height) for triplicate analysis measured with our prototype and with a commercial CE instrument, calculated using Chromophoreasy …”

Section: Resultsmentioning

confidence: 99%

Automated Capillary Electrophoresis System Compatible with Multiple Detectors for Potential In Situ Spaceflight Missions

et al. 2021

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The in situ search for chemical signatures of life on extraterrestrial worlds requires automated hardware capable of performing detailed compositional analysis during robotic missions of exploration. The use of electrophoretic separations in this search is particularly powerful, enabling analysis of a wide range of soluble organic compounds potentially indicative of life, as well as inorganic compounds that can serve as indicators of habitability. However, to detect this broad range of compounds with a single electrophoresis instrument, a combination of different detection modes is required. For detection of any ionizable species, including organic compounds that do not participate in terrestrial biology (i.e., “unknown unknowns”), mass spectrometry (MS) is essential. Inorganic ions, or any dissolved charged species present, can be analyzed using capacitively coupled contactless conductivity detection (C4D). Additionally, for the trace analysis of compounds of key interest to astrobiology (particularly, amino acids), laser-induced fluorescence (LIF) detection holds unique promise, due to the fact that it has the highest demonstrated sensitivity of any form of detection. Here, we demonstrate a fully automated, portable capillary electrophoresis analyzer that is capable of all these modes of detection. The prototype system developed here addresses the three most significant challenges for doing electrophoretic separations: precise sample injection, HV isolation, and automation of all operational steps. These key challenges were successfully addressed with the use of custom-designed rotor-stator valves with optimized operational sequences incorporating gas purging steps, rinses, and HV application.

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Section: Resultsmentioning

confidence: 99%

Automated Capillary Electrophoresis System Compatible with Multiple Detectors for Potential In Situ Spaceflight Missions

et al. 2021

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“…All hardware was controlled using MicroManager . Images were processed using FIJI and field-flattened to correct for nonuniform illumination across the channel. , Separation metrics were determined using Chromophoreasy (see Supporting Information (SI) for details) …”

Section: Materials and Methodsmentioning

confidence: 99%

“…24,35 Separation metrics were determined using Chromophoreasy (see Supporting Information (SI) for details). 36 Device Fabrication and Operation. Polydimethylsiloxane (PDMS) microchips were fabricated in-house using standard soft lithography methods.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Simultaneous Preconcentration and Separation of Native Protein Variants Using Thermal Gel Electrophoresis

et al. 2020

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Proteins must maintain proper folding conformations and express the correct post-translational modifications (PTMs) to exhibit appropriate biological activity. However, assessing protein folding and PTMs is difficult because routine polyacrylamide gel electrophoresis (PAGE) methods lack the separation resolution necessary to identify variants of a single protein. Additionally, standard PAGE denatures proteins prior to analysis precluding determinations of folding states or PTMs. To overcome these limitations, a microfluidic thermal gel electrophoresis platform was developed to provide high-sensitivity, high-resolution analyses of native protein variants. A thermally reversible gel was utilized as a separation matrix while in its solid state (30 °C). This thermal gel provided sufficient separation resolution to identify three variants of a fluorescently labeled model protein. To increase detection sensitivity, analyte preconcentration was conducted in parallel with the separation. Continuous analyte enrichment afforded detection limits of 500 fg of protein (250 pM) while simultaneous baseline separation resolution was achieved between variants. The effects of temperature on thermal gel electrophoresis were also characterized. The unique temperature-dependent outcomes illustrated how method performance can be tuned through a thermal dimension. Ultimately, the high detection sensitivity and separation resolution provided by thermal gel electrophoresis enabled rapid screening of native protein variants.

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“…Electropherograms were input into Chromophoreasy to calculate separation resolutions between proteins based on width at base (35). Separation resolutions were only calculated for analytes within a single domain (i.e.…”

Section: Analysis and Data Processingmentioning

confidence: 99%

Controlling the Separation of Native Proteins With Temperature in Thermal Gel Transient Isotachophoresis

Thanthri

Linz

2022

Preprint

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Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution. Thermal gels were employed as a separations matrix because of their unique ability to change viscosity in response to temperature. Proteins (6–464 kDa) were added into thermal gel and loaded into a microfluidic device. Electrolyte optimization was conducted to achieve robust tITP to isotachophoretically preconcentrate proteins and then electrophoretically separate them. Electropherograms were collected through both time and distance to enable both small and large proteins to be measured within a single analysis. The effects of temperature were evaluated and found to exhibit a pronounced effect on the separation. Temperature gradients were then employed to alter thermal gel viscosity over time to maximize separation resolution between proteins. The results herein demonstrate how gradient TG-tITP achieves rapid, high-resolution separations of native proteins over a wide mass range while requiring significantly less protein loading than PAGE and providing faster analysis times.

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Chromophoreasy, an Excel-Based Program for Detection and Integration of Peaks from Chromatographic and Electromigration Techniques

Cited by 9 publications

References 27 publications

Automated Capillary Electrophoresis System Compatible with Multiple Detectors for Potential In Situ Spaceflight Missions

Automated Capillary Electrophoresis System Compatible with Multiple Detectors for Potential In Situ Spaceflight Missions

Simultaneous Preconcentration and Separation of Native Protein Variants Using Thermal Gel Electrophoresis

Controlling the Separation of Native Proteins With Temperature in Thermal Gel Transient Isotachophoresis

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