ULTRAVIOLET MATRIX-ASSISTED LASER-DESORPTION IONIZATION-MASS SPECTROMETRY OF ELECTROBLOTTED PROTEINS

Direct mass spectrometric analysis of proteins electroblotted onto pol yvinylidene fluoride membranes after sodium dodecyl sulfate-polyacryla mide gel electrophoresis is demonstrated by matrix-assisted laser deso rption ionization-mass spectrometry (MALDI-MS) with a linear time-of-f light instrument, equipped with a nitrogen laser (337 nm). The blotted proteins were desorbed directly from the blotting membrane after incu bation with sinapinic acid as matrix. Different commercially available membranes resulted in high quality protein signals for hydrophobic me mbranes exhibiting high specific surface areas (Immobilon PSQ or Trans -Blot) of for charged membranes (Immobilon CD). Systematic investigati ons with standard proteins were performed to compare standard preparat ion procedures for ultraviolet (UV) MALDI-MS on stainless steel sample stages and preparation of proteins immobilized onto membranes either by direct application from protein solutions (spotting) or by electrot ransfer from gels (electroblotting). Aspects such as mass resolution, reproducibility from shot to shot and spot to spot, mass accuracy, and preservation of protein localization are addressed in this paper.