SILAC/TMT analysis of proteome expression in HeLa Cas9 and SR KO cells. (A) Schematic representation of combined SILAC/TMT multiplexed proteomic analysis. HeLa Cas9 WT and SRB KO2 cells were cultured in light and heavy amino acid culture media, respectively, for 7 d. On day 3, parental and SRB KO2 cells were transfected with nontargeting or SRA siRNA, respectively. Ninety-six hours post-transfection, equal numbers of light and heavy amino acid labeled cells were combined and homogenized. Homogenates were then centrifuged to separate the microsome/membrane pellet from the cytosol fraction. Membrane pellets were rehomogenized in carbonate/urea buffer and centrifuged to separate the integral membrane and peripheral membrane protein fractions. Biological replicate fractionation experiments were performed, fractions processed, subject to isobaric labeling (tandem mass tag, TMT), combined, and analyzed by LC–MS/MS. (B) Heat map plots illustrating compositions, reproducibility, and efficacy of cell fractionation protocol. Hierarchical clustering performed with scaling prior to clustering. (Mem) Membrane fraction, (Ext) urea/carbonate extractable membrane fraction, (Cyt) supernatant fraction after separation of membrane and membrane-associated proteins. (C) Volcano plot depicting log2-fold scaled protein distributions, comparing heavy and light amino acid labeled samples collected as in (A). (D) Schematic illustration of peptide coverage for SRA and SRB in HeLa Cas9 WT and SR KO cells, derived from TMT quantitation data sets. (E) Comparison of calculated protein abundances (spectral peptide index, SPI) determined as in (A), see also Materials and Methods, for proteins detected in the membrane fraction of HeLa Cas9 WT cells transfected with nontargeting siRNA and HeLa Cas9 SRB KO2 cells transfected with SRA siRNA, as described in (A). (Red) Integral membrane protein; (blue) other, nonmembrane protein; (yellow) ER resident integral membrane protein. A subset of ER-resident membrane protein distributions is indicated, as is the SRP54 subunit of SRP. (F) Table of relative log10 abundances of a subset of ER-resident membrane proteins, illustrating down-regulation of EMC4,9,6, and SRB/SRA subunits. See also Supplemental Data S1.
