SENSITIVITY AND MASS ACCURACY FOR PROTEINS ANALYZED DIRECTLY FROM POLYACRYLAMIDE GELS - IMPLICATIONS FOR PROTEOME MAPPING

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemo globin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsi nogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix wa s deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing tec hniques were capable of ameliorating some of the mass accuracy limitat ions inherent in desorbing from uneven insulator surfaces with externa l calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal g old staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automa tion.