Peptide separation with immobilized pI strips is an attractive alternative to in‐gel protein digestion for proteome analysis

Abstract

Complex protein mixtures have traditionally been separated by 2‐DE. Görg introduced IPGs as the first dimension of protein separation. In recent years, MS‐based proteomics has increasingly become the method of choice for identifying and quantifying large number of proteins. In that technology, to decrease analyte complexity, proteins are often separated by 1‐D SDS‐gel electrophoresis before online MS analysis. Here, we investigate a recently introduced device for peptide separation with IPGs (Agilent OFFGEL). Loading capacity for optimal peptide focusing is below 100 μg and – similar to 2‐D gels – IEF is more efficient in the acidic than the basic pH region. The 24‐well fractionation format resulted in about 40% additional peptide identifications but less than 20% additional protein identifications than the 12‐well format. Compared to in‐gel digestion, peptide IEF consistently identified a third more proteins with equal number of fractions. Low protein starting amounts (10 μg) still resulted in deep proteome coverage. Advantages of the in‐gel format include better reliability and robustness. Considering its superior performance, diminished sample and work‐up requirements, peptide IEF will become a method of choice for sample preparation in proteomics.