Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis

摘要

Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI–TOF/TOF)- basedlabel-freerelativeproteinquantificationstrategythat involves sodiumdodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) separationof proteins followedbyin-gel trypsindigestion. Themain problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT). The PCT approach performed at least as well as the conventionalovernight in-gel trypsindigestionapproachinparameters suchasnumberofpeaksdetected, number of peptides identified, and sequence coverage, and the digestion timewasreduced to 45 min. The gel/ mass spectrometry (MS)-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the liquid chromatography (LC)/MS platform. By normalizing the average signal intensities of the threemost intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides, which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10 and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins comigrating during electrophoretic separation.