TY - JOUR
T1 - Rapid LC-MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins
AU - Lippens, Jennifer L.
AU - Egea, Pascal F.
AU - Spahr, Chris
AU - Vaish, Amit
AU - Keener, James E.
AU - Marty, Michael T.
AU - Loo, Joseph A.
AU - Campuzano, Iain D.G.
N1 - Funding Information:
Support from the US National Institutes of Health (R01GM103479) and the US Department of Energy (UCLA/DOE Institute for Genomics and Proteomics; DE-FC03-02ER63421) to J.A.L. is acknowledged. M.T.M. and J.E.K. are supported by the Bisgrove Scholar Award from the Science Foundation Arizona and the National Institute of General Medical Sciences and National Institutes of Health under Award R35 GM128624 to M.T.M. The authors would like to acknowledge Drs. James Bowie and Nicholas Woodall (UCLA) for their generous gift of the bacteriorhodopsin constructs and Hui S. Tsui and Dr. Catherine F. Clarke (UCLA) for the gift of the Coq10 protein. Furthermore, the authors thank Tawnya Flick (Amgen) and Anthony Reed (Amgen) for the initial discussions regarding the analysis of bispecific antibody constructs and N-terminal formylation, respectively. Patrick Hoffmann (Amgen ARM Research and Development) and Will Hamouda (Amgen Pre-Pivotal Attribute Sciences) are acknowledged for providing the bispecific antibody constructs. Furthermore, the authors thank Mengjie Lu and Qiang Zhao (SIMM) and Dandan Zhang and Yingli Ma (Amgen Shanghai) for their willingness to provide additional membrane proteins for method testing. The Amgen postdoctoral program is acknowledged for its support of this project.
Publisher Copyright:
© 2018 American Chemical Society.
PY - 2018/11/20
Y1 - 2018/11/20
N2 - 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.
AB - 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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U2 - 10.1021/acs.analchem.8b03843
DO - 10.1021/acs.analchem.8b03843
M3 - Article
C2 - 30335969
AN - SCOPUS:85056703227
VL - 90
SP - 13616
EP - 13623
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 22
ER -