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Absolute quantification of proteins in the fatty acid biosynthetic pathway using protein standard absolute quantification.

ABSTRACT: With worldwide attention on renewable energy and climate change, metabolic engineering of the fatty acid biosynthetic pathway has become an active area of research, with a view to enhance production of biofuels. Indeed, this pathway has already been extensively studied in Escherichia coli. Nevertheless, little is known about the absolute abundance of the enzymes involved, information that may be valuable for engineering, such as the optimal molar ratios of different proteins. In this study, we use protein standard absolute quantification (PSAQ) to measure the absolute abundance of proteins that catalyze fatty acid biosynthesis in E. coli. In addition, the changes of protein abundance were analyzed by comparing the differences between high-yield and the background strain. Our work highlights opportunities to enhance fatty acid production by measuring protein molar ratios and identifying catalytic and regulatory bottlenecks. More importantly, our results provide evidence that PSAQ is a generally valuable tool to investigate metabolic pathways.

SUBMITTER: Tao H

PROVIDER: S-EPMC5640790 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Absolute quantification of proteins in the fatty acid biosynthetic pathway using protein standard absolute quantification.

Tao Hui H Zhang Yuchen Y Cao Xiaoying X Deng Zixin Z Liu Tiangang T

Synthetic and systems biotechnology 20160217 3

With worldwide attention on renewable energy and climate change, metabolic engineering of the fatty acid biosynthetic pathway has become an active area of research, with a view to enhance production of biofuels. Indeed, this pathway has already been extensively studied in <i>Escherichia coli.</i> Nevertheless, little is known about the absolute abundance of the enzymes involved, information that may be valuable for engineering, such as the optimal molar ratios of different proteins. In this stud ...[more]

PMID: 29062939

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Project description:The rapidly developing field of oncolytic virus (OV) therapy necessitates the development of new and improved analytical approaches for the characterization of the virus during production and development. Accurate monitoring and absolute quantification of viral proteins are crucial for OV product characterization and can facilitate the understanding of infection, immunogenicity, and development stages of viral replication. Targeted mass spectrometry methods like multiple reaction monitoring (MRM) offer a robust way to directly detect and quantify specific targeted proteins represented by surrogate peptides. We have leveraged the power of MRM by combining ultra-high performance liquid chromatography (UPLC) with a Sciex 6500 triple-stage quadrupole mass spectrometer to develop an assay that accurately and absolutely quantifies the structural proteins of a pseudotyped vesicular stomatitis virus (VSV) intended for use as a new biotherapeutic (designated hereafter as VSV-GP to differentiate it from native VSV). The new UPLC-MRM method provides absolute quantification with the use of heavy-labeled reference standard surrogate peptides. When added in known exact amounts to standards and samples, the reference standards normalize and account for any small perturbations during sample preparation and/or instrument performance, resulting in accurate and precise quantification. Because of the multiplexed nature of MRM, all targeted proteins are quantified at the same time. The optimized assay has been enhanced to quantify the ratios of the processed GP1 and GP2 proteins while simultaneously measuring any remaining or unprocessed form of the envelope protein GP complex (GPC; full-length GPC).ImportanceThe development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (VSV-GP) is a new biotherapeutic emerging in the oncolytic viral therapy platform. Novel analytical assays that can accurately and precisely quantify the viral proteins are a necessity for the successful development of viral vector as a biotherapeutic. We developed an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the infectivity of the virus. The assay extends the potential for quantifying full-length GPC, which provides an understanding of the processing of GPC (along with the quantification of GP1 and GP2 separately). We used this assay in tracking GPC processing in HEK-293-F production cell lines infected with VSV-GP.

Dataset Information

Absolute quantification of proteins in the fatty acid biosynthetic pathway using protein standard absolute quantification.

Publications

Absolute quantification of proteins in the fatty acid biosynthetic pathway using protein standard absolute quantification.

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