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An improved fast photochemical oxidation of proteins (FPOP) platform for protein therapeutics.

ABSTRACT: Unlike small-molecule drugs, the size and dynamics of protein therapeutics challenge existing methods for assessing their high order structures (HOS). To extend fast photochemical oxidation of proteins (FPOP) to protein therapeutics, we modified its platform by introducing a mixing step prior to laser irradiation to minimize unwanted H(2)O(2)-induced oxidation. This improvement plus standardizing each step yield better reproducibility as determined by a fitting process whereby we used a non-FPOP spectrum as a template to report the unmodified level. We also tested different buffer systems for this modified FPOP platform with cytochrome c. The outcome is a standard oxidation profile that can be compared between different laboratories and regulatory agencies that wish to adopt FPOP for quality control purposes.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC5993200 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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An improved fast photochemical oxidation of proteins (FPOP) platform for protein therapeutics.

Zhang Ying Y Rempel Don L DL Zhang Hao H Gross Michael L ML

Journal of the American Society for Mass Spectrometry 20141218 3

Unlike small-molecule drugs, the size and dynamics of protein therapeutics challenge existing methods for assessing their high order structures (HOS). To extend fast photochemical oxidation of proteins (FPOP) to protein therapeutics, we modified its platform by introducing a mixing step prior to laser irradiation to minimize unwanted H(2)O(2)-induced oxidation. This improvement plus standardizing each step yield better reproducibility as determined by a fitting process whereby we used a non-FPOP ...[more]

PMID: 25519854

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Project description:Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

Dataset Information

An improved fast photochemical oxidation of proteins (FPOP) platform for protein therapeutics.

Publications

An improved fast photochemical oxidation of proteins (FPOP) platform for protein therapeutics.

Similar Datasets

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