Obtaining Molecular Weights of Proteins and Their Cleavage Products by Directly Combining Gel Electrophoresis with Mass Spectrometry

Methods combining the high throughput and sensitivity of matrix-assisted laser desorption ionization mass spectometry (MALDI-MS) with polyacrylamide gel electrophoresis (PAGE) are rapidly gaining attention, but almost exclusively for applications eluting proteolytic digest products from gels and membranes (1 –9 ). These methods fulfill a considerable need for identifying unknown proteins isolated in polyacrylamide gels, but they do not provide masses for intact proteins, a particular concern should the goal be the understanding of anomalous electrophoretic migrations, characterizing the complete ensemble of posttranslational modifications displayed by proteins, or understanding why multiple protein spots on a two-dimensional (2-D) gel are all identified as the same protein. These issues are particularly relevant for low-abundance proteins, which may be identified by matching the masses of only a few tryptic peptides to those predicted from cleavage of an unmodified protein, or by matching small stretches of sequence information (9 ,10 ). In some cases, those matches may correspond to <10% of the total protein sequence (9 ). Analysis of intact proteins has been performed following electroblotting to membranes (11 –19 ), but we will describe a method to link gel electrophoresis directly to MALDI-MS, yielding masses of both intact proteins and cleavage products without electroelution or electroblotting (20 –22 ). Key to the success of this approach are ultrathin polyacrylamide gels, which dry to thicknesses of 10 μm or less and which have the additional advantages of rapid preparation and electrophoresis run times.