1 research outputs found
Tag and Capture Flow Hydrogen Exchange Mass Spectrometry with a Fluorous-Immobilized Probe
Analysis of complex mixtures of proteins
by hydrogen exchange (HX)
mass spectrometry (MS) is limited by one’s ability to resolve
the protein(s) of interest from the proteins that are not of interest.
One strategy for overcoming this problem is to tag the target protein(s)
to allow for rapid removal from the mixture for subsequent analysis.
Here we illustrate a new solution involving fluorous conjugation of
a retrievable probe. The appended fluorous tag allows for facile immobilization
on a fluorous surface. When a target protein is passed over the immobilized
probe molecule, it can be efficiently captured and then exposed to
a flowing stream of deuterated buffer for hydrogen exchange. The utility
of this method is illustrated for a model system of the Elongin BC
protein complex bound to a peptide from HIV Vif. Efficient capture
is demonstrated, and deuteration when immobilized was identical to
deuteration in conventional solution-phase hydrogen exchange MS. Protein
captured from a crude bacterial cell lysate could also be deuterated
without the need for separate purification steps before HX MS. The
advantages and disadvantages of the method are discussed in light
of miniaturization and automation