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. 2022 Mar 8;11:e73982. doi: 10.7554/eLife.73982

Figure 3. WGA-HRP identifies a number of enriched markers on Myc-driven prostate cancer cells.

(A) Overall scheme for biotin labeling, and label-free quantification (LFQ) by LC-MS/MS for RWPE-1 Control and Myc overexpression cells. (B) Microscopy image depicting morphological differences between RWPE-1 Control and RWPE-1 Myc cells after 3 days in culture. (C) Volcano plot depicting the LFQ comparison of RWPE-1 Control and Myc labeled cells. Red labels indicate upregulated proteins in the RWPE-1 Control cells over Myc cells and green labels indicate upregulated proteins in the RWPE-1 Myc cells over Control cells. All colored proteins are at least two-fold enriched in either dataset between four replicates (two technical, two biological, p<0.05). (D) Heatmap of the 30 most upregulated transmembrane (bold) or secreted (italics) proteins in either RWPE-1 Control or Myc cells. Scale indicates intensity, defined as (LFQ Area−Mean LFQ Area)/Standard Deviation. (E) Table indicating fold-change of most differentially regulated proteins by LC-MS/MS for RWPE-1 Control and Myc cells. (F) Upregulated proteins in RWPE-1 Myc cells (Myc, ANPEP, Vimentin, and FN1) are confirmed by western blot. (G) Upregulated surface proteins in RWPE-1 Myc cells (Vimentin, ANPEP, and FN1) are detected by immunofluorescence microscopy. The downregulated protein HLA-B by Myc overexpression was also detected by immunofluorescence microscopy. All western blot images and microscopy images are representative of two biological replicates. Mass spectrometry data is based on two biological and two technical replicates (N=4).

Figure 3—source data 1. Uncropped western blots.
Figure 3—source data 2. Mass spectrometry analysis results table.
elife-73982-fig3-data2.xlsx (688.1KB, xlsx)
Figure 3—source data 3. List of proteins comparing enriched targets (>2-fold) in Myc cells versus Control cells.

Figure 3.

Figure 3—figure supplement 1. Comparison of surface enrichment between replicates for different mass spectrometry methods.

Figure 3—figure supplement 1.

(A) The top three methods (NHS-Biotin, Biocytin Hydrazide, and WGA-HRP) were compared for their ability to enrich cell surface proteins on 1.5 M RWPE-1 Control cells by LC-MS/MS after being searched with the Uniprot GOCC Plasma Membrane database. Shown are enrichment levels on the protein, peptide, and average MS1 intensity of top three peptides (LFQ area) levels. (B) The top three methods (NHS-Biotin, Biocytin Hydrazide, and WGA-HRP) were compared for their ability to enrich cell surface proteins on 1.5 M RWPE-1 Control cells by LC-MS/MS after being searched with the entire human Uniprot database. Shown are enrichment levels on the protein, peptide, and average MS1 intensity of top three peptides (LFQ area) levels. Proteins or peptides detected from cell surface annotated proteins (determined by the SURFY database) were divided by the total number of proteins or peptides detected. LFQ areas corresponding to cell surface annotated proteins (SURFY) were divided by the total area sum intensity for each sample. The corresponding percentages for two biological replicates were plotted. LFQ, label-free quantification.
Figure 3—figure supplement 1—source data 1. Mass spectrometry analysis results.
Figure 3—figure supplement 2. Comparison of replicates for different mass spectrometry methods shows that WGA-HRP has comparable reproducibility to NHS-Biotin and Hydrazide labeling.

Figure 3—figure supplement 2.

(A) Spearman correlations of total area sum intensity normalized data from replicates of Hydrazide Control and Myc cells. (B) Spearman correlations of total area sum intensity normalized data from replicates of NHS Control and Myc cells. (C) Spearman correlations of total area sum intensity normalized data from replicates of WGA Control and Myc cells.