2-d clean-up  (Verlag GmbH)

Journal: Oncogene

Article Title: EphA2 regulates vascular permeability and prostate cancer metastasis via modulation of cell junction protein phosphorylation

doi: 10.1038/s41388-024-03206-x

Figure Lengend Snippet: a Schematic representation of phosphoproteomic workflow. PC-3 cells were differentially labeled with light ( 12 C) and heavy ( 13 C) Arg and Lys isotopes for 6 passages, then activated with clustered ephrin-A1-Fc (efnA1-Fc) or treated with clustered Fc control for 20 min. Following cell lysis and clean-up, equal amounts of lysates from each condition were combined and digested with LysC and trypsin. Phosphopeptides were enriched with TiO 2 and analyzed by LC-MS/MS. N = 4 independently SILAC-labeled, stimulated and processed biological replicates. The light and heavy amino acid labeling was alternated between replicates (“label swap”): replicates 1 and 3 were “forward labeled” (Fc control: light, ephrin-A1-Fc: heavy) and replicates 2 and 4 were “reverse labeled”. b Heat map showing the log2-transformed activated:control ratios across the four replicates for the 30 most regulated phosphopeptides. c Regulated phosphoproteins were categorized into protein classes according to function by manually curating information from HRPD, Panther and UniProt databases and the literature. GO enrichment analysis of Cellular Compartment ( d ) and Biological Process and Molecular Function terms ( e ). The ClueGo app for Cytoscape was used to calculate enrichment of terms associated with EphA2-regulated proteins compared to our own background dataset and to cluster related and redundant terms. Enrichment was evaluated using one-sided hypergeometric testing. FDR was controlled using the Benjamini–Hochberg correction for multiple hypothesis testing. Node size reflects the number of regulated phosphoproteins linked to a particular term; node color indicates the Benjamini–Hochberg corrected p value as a measure of significance as indicated.

Article Snippet: Equal amounts (800 μg) of cell lysates from “heavy” and “light” conditions were pooled and proteins enriched using a 2D clean-up kit (GE Healthcare) and quantitated using the 2D Quant kit (GE Healthcare).

Techniques: Labeling, Control, Lysis, Liquid Chromatography with Mass Spectroscopy, Multiplex sample analysis, Transformation Assay