A Novel Prefractionation Method Combining Protein and Peptide Isoelectric Focusing in Immobilized pH Gradient Strips

To increase sensitivity and analytical depth in shotgun
proteomics, prefractionation of complex samples is often used. Here
we describe a novel prefractionation method, Sandwich high resolution
isoelectric focusing, which combines both protein and peptide isoelectric
focusing. In the first step, intact proteins are separated on the
basis of isoelectric point (pI) using traditional
immobilized pH gradient (IPG) strips. Segments in the IPG-strip containing
proteins of interest are subsequently cut out and applied to in-strip
digestion, without subsequent peptide elution. In the second peptide isoelectric focusing step, the strip segments are
used as loading bridges. The peptides are thereby directly applied
to the peptide isoelectric focusing, without an intermediate elution
step, and separated on narrow range IPG strips to reduce the complexity
on the peptide level. In the final step, the peptides are eluted into
96-well plates and analyzed with mass spectrometry. In a proof of
principle experiment, using this method to zoom in on pI regions of interest in human plasma, we identify over 800 proteins,
with concentrations spanning over 6 orders of magnitude.