Discovery of Manassantin A Protein Targets Using Large-Scale Protein Folding and Stability Measurements

Abstract

Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A's mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein-ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action.

Keywords: Filamin A; MDA-MB-231; Manassantin A; SILAC; SPROX; cancer; chemical denaturation; elongation factor 1α; iTRAQ; mass spectrometry; mode-of-action; proteomics; pulse proteolysis.

Figure 1

Schematic representation of the iTRAQ-SPROX workflow utilized in this work.

Figure 2

Representative iTRAQ-SPROX data sets. Chemical denaturation data generated for a non-hit peptide TLTIVDTGIGMTK from Hsp90 assayed in Exp. 1 is shown in (A). The data for two peptide hits, MDSTEPPYSQK and GAGSYTIMVLFADQATPTSPIR, from elongation factor 1α and filamin A are shown in (B) and (C) (respectively). The light and dark shaded bars correspond to the (−) and (+) The C_1/2 values are indicated with vertical arrows. The horizontal dashed lines indicate the normalized reporter ion intensity that was used as the cut-off value that separates the pre- and post-transition baselines. In cases where data from multiple product ion mass spectra was collected, the averaged data is reported along with error bars that represent one standard deviation.

Figure 3

Schematic representation of the SILAC-PP workflow utilized in this work.

Figure 4

SILAC-PP data generated on three iTRAQ-SPROX protein hits. (A) The average Log₂ L/H ratios determined from 15 filamin A peptides in the analysis of the gel-bands excised from row 1 in the SDS-PAGE gel (i.e., the >150 kDa region of the gel). (B) The Log2 L/H ratios determined for the a peptide from elongation factor 1α in the analysis of the gel bands excised from row 5 in the SDS-PAGE gel (i.e., the 55–65 kDa region of the gel). The horizontal dotted lines mark the regions within 1.7-fold of the median L/H ratio of all the peptides identified in the SILAC-PP experiment

Figure 5

Schematic representation of filamin A. The arrows indicate the location of the 13 methionine residues in the 13 methionine-containing peptide probes assayed in the iTRAQ-SPROX experiment described here. The dotted arrow indicates the location of the methionine in the hit peptide probe.