Supplementary Figure 2: Fractionation of human whole-cell lysate prior to top-down mass spectrometry.
From: Best practices and benchmarks for intact protein analysis for top-down mass spectrometry
Human colorectal cancer cells were lysed and constituent proteins quantified by the methods described by Anderson et. al.1 Aliquots of lysate containing 400 µg were precipitated in acetone, resuspended in 1% SDS containing 50 mM DTT, and resolved on 8% T (a.) or 10% T (b.) gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) cartridges following the respective manufacturer’s protocols (GELFrEE 8100 Fractionation System, Expedeon, Inc.). Upon collection of MW-based fractions, 10 µL aliquots were resolved by SDS-PAGE and visualized by AgNO3 stain2 to gauge protein content and quality of resolution. (a,b) Note that the MW ranges of f1 (purple box) and subsequent fractions differ depending on the GELFrEE cartridge selected. While 8% cartridges (a.) are recommended for quantitative high-throughput top-down MS applications or analysis of higher-MW proteins, 10% (b.) cartridges provide superior resolution in the 5-30 kDa MW range for qualitative high-throughput applications. 1. Anderson, L.C. et al. Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry. J Proteome Res 16, 1087-1096 (2017). 2. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850-858 (1996).
