Rapid LC–MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins
| Autor: | Iain D. G. Campuzano, Pascal F. Egea, James E. Keener, Amit Vaish, Michael T. Marty, Joseph A. Loo, Jennifer L. Lippens, Chris Spahr |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Bispecific antibody Chromatography Chemistry 010401 analytical chemistry Membrane Proteins Mass spectrometry 01 natural sciences Article Mass Spectrometry 0104 chemical sciences Analytical Chemistry Molecular Weight 03 medical and health sciences 030104 developmental biology Membrane Membrane protein Liquid chromatography–mass spectrometry Hydrophobic and Hydrophilic Interactions Ion channel Chromatography Liquid G protein-coupled receptor |
| Zdroj: | Analytical Chemistry. 90:13616-13623 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | 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 development of a 5-minute 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-minute method detailed has successfully produced MW measurements for hydrophobic proteins with a wide size range (17.5 to 105 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 non-formylated aquaporinZ membrane protein. |
| Databáze: | OpenAIRE |
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