posted on 2018-10-17, 00:00authored byJennifer L. Lippens, Pascal F. Egea, Chris Spahr, Amit Vaish, James E. Keener, Michael T. Marty, Joseph A. Loo, Iain D. G. Campuzano
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.