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 α‐ and β‐chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was 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 techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa α‐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 gold 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 automation.

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