Resolubilization of Precipitated Intact Membrane Proteins with Cold Formic Acid for Analysis by Mass Spectrometry

Protein precipitation in organic
solvent is an effective strategy
to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here
we evaluate the recovery of membrane and water-soluble proteins through
precipitation with chloroform/methanol/water or with acetone (80%).
With each solvent system, membrane protein recovery was greater than
90%, which was generally higher than that of cytosolic proteins. With
few exceptions, residual supernatant proteins detected by MS were
also detected in the precipitation pellet, having higher MS signal
intensity in the pellet fraction. Following precipitation, we present
a novel strategy for the quantitative resolubilization of proteins
in an MS-compatible solvent system. The pellet is incubated at −20
°C in 80% formic acid/water and then diluted 10-fold with water.
Membrane protein recovery matches that of sonication of the pellet
in 1% SDS. The resolubilized proteins are stable at room temperature,
with no observed formylation as is typical of proteins suspended in
formic acid at room temperature. The protocol is applied to the molecular
weight determination of membrane proteins from a GELFrEE-fractionated
sample of Escherichia coli proteins.