Abstract
In proteomics, one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein fractionation prior to mass spectrometric analysis to enhance the dynamic range of analysis and to improve the identification of low-abundance proteins. Such protein prefractionation works well for quantitation strategies if the proteins are labeled prior to separation. However, because of the poor reproducibility of cutting gel slices, especially when small amounts of samples are analyzed, its application in label-free and peptide-labeling quantitative proteomics methods has been greatly limited. To overcome this limitation, we developed a new strategy in which a DNA ladder is mixed with the protein sample before PAGE separation. After PAGE separation, the DNA ladder is stained to allow for easy, precise, and reproducible gel cutting. To this end, a novel visible DNA-staining method was developed. This staining method is fast, sensitive, and compatible with mass spectrometry. To evaluate the reproducibility of DNA-ladder-assisted gel cutting for quantitative protein fractionation, we used stable isotope labeling with amino acids in cell culture (SILAC). Our results show that the quantitative error associated with fractionation can be minimized using the DNA-assisted fractionation and multiple replicates of gel cutting. In conclusion, 1D PAGE fractionation in combination with DNA ladders can be used for label-free comparative proteomics without compromising quantitation.