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 - 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.
UR - http://www.scopus.com/inward/record.url?scp=85056703227&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85056703227&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.8b03843
DO - 10.1021/acs.analchem.8b03843
M3 - Article
C2 - 30335969
AN - SCOPUS:85056703227
SN - 0003-2700
VL - 90
SP - 13616
EP - 13623
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 22
ER -