High Sensitivity Protein Gel Electrophoresis Label Compatible with Mass-Spectrometry

Abstract

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for macromolecule and protein analysis. While multiple methods exist to visualize the separated protein bands on gels, one of most popular methods of staining the proteins is with Coomassie dye. A more recent approach is to use Bio-Rad stain-free technology for visualizing protein bands with UV light and achieve similar or greater sensitivity than that of Coomassie dye. Here, we developed a method to further amplify the sensitivity of stain-free gels using carboxyfluorescein succinimidyl ester (CFSE) staining. We compared our novel method using foetal bovine serum samples with Coomassie dye, standard stain-free gels, and silver staining. Our results show that while silver staining remains a gold-standard method in terms of sensitivity; CFSE staining of samples prior to use with stain-free gels results in a 10-100-fold increase in sensitivity over Coomassie staining and the standard stain-free method. Our method offers a sensitivity similar to that of silver staining which is compatible with downstream mass spectrometry, and therefore more advantageous for further retrieval and analysis of macromolecules in bands.

Keywords: extracellular vesicles; gel electrophoreses; labelling; mass spectrometry; protein.

Figure 1

NanoDrop protein concentration approximation using serially diluted fetal bovine serum (FBS). Regression was performed with data from serially diluted FBS, with three measurements shown per dilution.

Figure 2

The effect of standard reduction methods upon carboxyfluorescein succinimidyl ester (CFSE)-labeled samples. Standard reduction methods include 95 °C for 10 min and 2-mercaptoethanol used alone and in combination with CFSE-stained and unstained samples on an SDS-PAGE stain-free gel.

Figure 3

Sensitivity comparison of silver stain, Coomassie blue, and stain-free methodologies with the same loaded FBS samples with and without CFSE staining.

Figure 4

Mass spectrometry data comparison of CFSE stained and unstained samples. Proteins were in-gel trypsin digested and analyzed by tandem mass spectrometry. Scatter diagrams. (A) The number of unique peptides detected per protein from stained vs. unstained samples; (B) Ratio of unique peptides in the CFSE stained over unstained vs. molecular weight; (C) Number of peptide spectra matches per protein in the stained vs. unstained samples; (D) Ratio of unique peptides in the CFSE stained over unstained vs. isoelectric point.