RTI uses cookies to offer you the best experience online. By clicking “accept” on this website, you opt in and you agree to the use of cookies. If you would like to know more about how RTI uses cookies and how to manage them please view our Privacy Policy here. You can “opt out” or change your mind by visiting: http://optout.aboutads.info/. Click “accept” to agree.
Shotgun proteomics, where a tryptic digest of a complex proteome sample is directly analyzed by either single dimensional or multidimensional liquid chromatography tandem mass spectrometry, has gained acceptance in the proteomics community at large and is widely used in core facilities. Here we review the development in our laboratory of an alternative first-dimension separation technique for shotgun proteomics, immobilized pH gradient isoelectric focusing (IPG-IEF). The key advantages of the technology over other multidimensional separation formats (simplicity, high resolution, and high sensitivity) are discussed. The concept of using peptide pI to filter large shotgun proteomics datasets generated by the IPG-IEF technique to minimize false positives and negatives is also introduced. Finally, an account of the comparison of the technique with the established gold standard for multidimensional separation of peptides, strong cation exchange chromatography, is presented, along with the prospects for the use of peptide pI along with accurate mass measurement for the identification of peptides