A comparison of immobilized pH gradient isoelectric focusing and strong‐cation‐exchange chromatography as a first dimension in shotgun proteomics

Abstract

Recently, we have developed a high‐resolution two‐dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed‐phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong‐cation‐exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first‐dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5–4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off‐line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5–4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false‐positives from the data.