期刊
ANALYTICAL CHEMISTRY
卷 90, 期 22, 页码 13616-13623出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.8b03843
关键词
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资金
- US National Institutes of Health [R01GM103479]
- US Department of Energy (UCLA/DOE Institute for Genomics and Proteomics) [DE-FC03-02ER63421]
- Science Foundation Arizona
- National Institute of General Medical Sciences
- National Institutes of Health [R35 GM128624]
- Amgen postdoctoral program
Therapeutic target characterization involves many components, including accurate molecular weight (MW) determination. Knowledge of the accurate MW allows one to detect the presence of post-translational modifications, proteolytic cleavages, and importantly, if the correct construct has been generated and purified. Denaturing liquid chromatography-mass spectrometry (LC-MS) can be an attractive method for obtaining this information. However, membrane protein LC-MS methodology has remained relatively under-explored and under-incorporated in comparison to methods for soluble proteins. Here, systematic investigation of multiple gradients and column chemistries has led to the development of a 5 min denaturing LC-MS method for acquiring membrane protein accurate MW measurements. Conditions were interrogated with membrane proteins, such as GPCRs and ion channels, as well as bispecific antibody constructs of variable sizes with the aim to provide the community with rapid LC-MS methods necessary to obtain chromatographic and accurate MW measurements in a medium to high-throughput manner. The 5 min method detailed has successfully produced MW measurements for hydrophobic proteins with a wide MW range (17.5 to 105.3 kDa) and provided evidence that some constructs indeed contain unexpected modifications or sequence clipping. This rapid LC-MS method is also capable of baseline separating formylated and nonformylated aquaporinZ membrane protein.
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