Mass spectrometry-based label free quantification of gel separated proteins.

Co-migrating proteins in 2D PAGE spots cause difficulties in the assignment of quantitative data obtained from the staining density of a gel spot. We present an application of a label free LC-MS quantitative method that can overcome problems like this. Protein mixtures were prepared with varying compositions, and were run on either 1D or 2D PAGE. Relative and absolute quantitative evaluation was carried out using a simple but reliable method based on integrated MS signals of the three most intensive peptides of each protein. The efficacy of digestion and peptide extraction is, however, influenced by different factors in gel from those in solution, hence the method had to be validated via a quantitative assessment of proteins from 1D or 2D gels. Our findings suggest that a reliable relative quantification is viable using peptide ion intensities when protein levels in two gels have to be compared. In the case of 2D gels, label free MS quantification provides more precise results on changes of protein expression levels than gel spot intensity-based measurements, especially in the case of overlapping proteins. Absolute amounts of different proteins in 1D or 2D gels can be evaluated to a reasonable precision.