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Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec Processing Pipeline (UPP) for the analysis of batched biotherapeutic intact MS data. UPP is built into the UniDec software package, which provides fast processing, deconvolution, and peak detection. The user and programming interfaces for UPP read a spreadsheet that contains the data file names, deconvolution parameters, and quantitation settings. After iterating through the spreadsheet and analyzing each file, it returns a spreadsheet of results and HTML reports. We demonstrate the use of UPP to measure correct pairing percentage on a set of bispecific antibody data and to measure drug-to-antibody ratios from antibody-drug conjugates. Moreover, because the software is free and open-source, users can easily build on this platform to create customized workflows and calculations. Thus, UPP provides a flexible workflow that can be deployed in diverse settings and for a wide range of biotherapeutic applications.
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DNA Analysis by Native Polyacrylamide Gel Electrophoresis and Infrared Fluorescence Imaging V.2
Short DNA probes, for example, those used in characterizing protein-DNA complexes, have historically been radiolabeled with 32P to allow their detection and quantitation following native polyacrylamide gel electrophoresis (PAGE). For reasons of economy or safety, alternative means of DNA detection need to be used. Described here is the resolution of 5' IRD700 and IRD800 end-labeled DNAs by native PAGE and their visualization and quantitation by IR fluorescence imaging.
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- Award ID(s):
- 1714778
- PAR ID:
- 10098104
- Date Published:
- Journal Name:
- Protocols.io
- ISSN:
- 2473-1838
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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High-Performance Liquid Chromatography (HPLC) and Gas Chromatography are analytical techniques which allow for the quantitative characterization of the chemical components of mixtures. Technological advancements in sample preparation and mechanical automation have allowed HPLC to become a high-throughput tool which poses new challenges for reproducible and rapid analysis of the resulting chromatograms. Here we present hplc-py, a Python package that permits rapid and reliable quantitation of component signals within a chromatogram for pipelined workflows. This is achieved by a signal detection and quantitation algorithm which i) identifies windows of time which contain peaks and ii) infers the parameters of a mixture of amplitude-weighted skew-normal distributions which sum to reconstruct the observed signal. This approach is particularly effective at deconvolving highly overlapping signals, allowing for precise absolute quantitation of chemical constituents with similar chromatographic retention times.more » « less
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Abstract As a highly successful introduced species, house sparrows (Passer domesticus) respond rapidly to their new habitats, generating phenotypic patterns across their introduced range that resemble variation in native regions. Epigenetic mechanisms likely facilitate the success of introduced house sparrows by aiding particular individuals to adjust their phenotypes plastically to novel conditions. Our objective here was to investigate patterns of DNA methylation among populations of house sparrows at a broad geographic scale that included different introduction histories: invading, established, and native. We defined the invading category as the locations with introductions less than 70 years ago and the established category as the locations with greater than 70 years since introduction. We screened DNA methylation among individuals (n = 45) by epiRADseq, expecting that variation in DNA methylation among individuals from invading populations would be higher when compared with individuals from established and native populations. Invading house sparrows had the highest variance in DNA methylation of all three groups, but established house sparrows also had higher variance than native ones. The highest number of differently methylated regions were detected between invading and native populations of house sparrow. Additionally, DNA methylation was negatively correlated to time-since introduction, which further suggests that DNA methylation had a role in the successful colonization’s of house sparrows.more » « less
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Abstract Capillary electrophoresis methods for the separation of carbohydrates with four different detection techniques, namely direct UV, indirect UV, capacitively coupled conductivity, and laser-induced fluorescence detection, were tested and their performance was evaluated and compared in terms of linearity, limits of detection and quantitation, repeatability, recovery, analysis time, and sample treatment. The test set of analytes comprised sucrose, glucose, and fructose. The effect of using lactose as an internal standard on the individual methods was investigated, too. The results showed that laser-induced fluorescence detection is a technique of choice for applications requiring the detection of very low amounts of reducing carbohydrates. Contactless conductivity detection is favorable when detection sensitivity is not a crucial parameter but fast and reliable analysis is required. When only a UV detector is available as a standard part of capillary electrophoresis instruments, direct UV detection can be used when analysis time is not a critical parameter. For fast analysis with UV detectors, indirect UV detection is the technique of choice. Finally, to verify the applicability of the tested methods, samples of cola beverage, honey, and orange juice were analyzed and the results obtained by all four methods were compared. Graphical abstractmore » « less
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The analysis of organic acids in complex mixtures by LC‐MS can often prove challenging, especially due to the poor sensitivity of negative ionization mode required for detection of these compounds in their native (i.e., underivatized or untagged) form. These compounds have also been difficult to measure using supercritical fluid chromatography (SFC)‐MS, a technique of growing importance for metabolomic analysis, with similar limitations based on negative ionization. In this report, the use of a high proton affinity N ‐(4‐aminophenyl)piperidine derivatization tag is explored for the improvement of organic acid detection by SFC‐MS. Four organic acids (lactic, succinic, malic, and citric acids) with varying numbers of carboxylate groups were derivatized with N ‐(4‐aminophenyl)piperidine to achieve detection limits down to 0.5 ppb, with overall improvements in detection limit ranging from 25‐to‐2100‐fold. The effect of the derivatization group on sensitivity, which increased by at least 200‐fold for compounds that were detectable in their native form, and mass spectrometric detection are also described. Preliminary investigations into the separation of these derivatized compounds identified multiple stationary phases that could be used for complete separation of all four compounds by SFC. This derivatization technique provides an improved approach for the analysis of organic acids by SFC‐MS, especially for those that are undetectable in their native form.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.17504/protocols.io.mcyc2xw
