In-Gel Isoelectric Focusing of Peptides as a Tool for Improved Protein Identification

In the analysis of proteins in complex samples, pre-fractionation is imperative to obtain the necessary
depth in the number of reliable protein identifications by mass spectrometry. Here we explore isoelectric
focusing of peptides (peptide IEF) as an effective fractionation step that at the same time provides the
added possibility to eliminate spurious peptide identifications by filtering for pI. Peptide IEF in IPG
strips is fast and sharply confines peptides to their pI. We have evaluated systematically the contribution
of pI filtering and accurate mass measurements on the total number of protein identifications in a
complex protein mixture (Drosophila nuclear extract). At the same time, by varying Mascot identification
cutoff scores, we have monitored the false positive rate among these identifications by searching reverse
protein databases. From mass spectrometric analyses at low mass accuracy using an LTQ ion trap,
false positive rates can be minimized by filtering of peptides not focusing at their expected pI. Analyses
using an LTQ-FT mass spectrometer delivers low false positive rates by itself due to the high mass
accuracy. In a direct comparison of peptide IEF with SDS-PAGE as a pre-fractionation step, IEF delivered
25% and 43% more proteins when identified using FT−MS and LTQ−MS, respectively. Cumulatively,
2190 non redundant proteins were identified in the Drosophila nuclear extract at a false positive rate
of 0.5%. Of these, 1751 proteins (80%) were identified after peptide IEF and FT-MS alone. Overall, we
show that peptide IEF allows to increase the confidence level of protein identifications, and is more
sensitive than SDS-PAGE.
Keywords: false positive rate • isoelectric focusing • SDS-PAGE • FT−MS • LTQ−MS • Drosophila