Observation of sodium gel-induced protein modifications in dodecylsulfate polyacrylamide gel electrophoresis and its implications for accurate molecular weight determination of gel-separated proteins by matrix-assisted laserdesorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spe ctrometry (TOFMS) can potentially provide accurate molecular weight informa tion of proteins separated by sodium dodecylsulfate polyacrylamide gel elec trophoresis (SDS-PAGE). Several issues related to resolution and accuracy o f molecular weight measurement are investigated by using a time-lag focusin g MALDI-TOF mass spectrometer. The effects of the gel components SDS, glyce rol, and tris buffer on the mass spectral signals are studied systematicall y. Glycerol and tris buffer are shown to have little or no effect on resolu tion and mass accuracy, whereas SDS degrades sensitivity, resolution, and m ass accuracy even at low concentrations. A simple and fast gel extraction t echnique is presented which is capable of detecting proteins loaded at the low-picomole level on the gel. The sample preparation procedure used in thi s work appears to remove most of SDS from the gel, thereby reducing the pea k broadening effect caused by SDS and resulting in high resolution and accu rate measurement of proteins. However, for proteins containing cysteines, t he molecular ions are composed of a distribution of acrylamide-protein addu cts likely formed by reaction with unpolymerized acrylamide in the gel duri ng the gel separation process. The implications of gel-induced protein modi fications on the accurate molecular weight measurement of gel-separated pro teins are discussed. (C) 1999 American Society for Mass Spectrometry.