Proteoform Catcher: The Power of 2D SDS-PAGE, Western Blotting, and Mass Spectrometry in Protein Characterization.

Abstract

Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) is a method that separates proteins according to their isoelectric point in the first dimension and molecular mass in the second dimension. Evidence is provided that 2D SDS-PAGE is reproducible, robust, and compatible with SDS in both dimensions, including isoelectric focusing in tube gels, the first dimension. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer, and is better yet when dissolved in a mixture of both buffers. Quantification of 60 proteins in rat liver cytosol over a wide range of pI and MW gave linear plots of spot density versus total protein for loads of 200, 400, and 600 µg protein dissolved in SDS buffer and run in triplicate on 2D gels (Average R² = 0.987). Examples are provided in which proteins found by 2D gel western blotting, cut from stained gels, and identified by mass spectrometry (MS) are proteoforms with unique post-translational modifications. Such proteins would not have been found by either method alone.