Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins Using SILAC-SPROX

Abstract

Proteomic methods for disease state characterization and biomarker discovery have traditionally utilized quantitative mass spectrometry methods to identify proteins with altered expression levels in disease states. Here we report on the large-scale use of protein folding stability measurements to characterize different subtypes of breast cancer using the stable isotope labeling with amino acids in cell culture and stability of proteins from rates of oxidation (SILAC-SPROX) technique. Protein folding stability differences were studied in a comparison of two luminal breast cancer subtypes, luminal-A and -B (i.e., MCF-7 and BT-474 cells, respectively), and in a comparison of a luminal-A and basal subtype of the disease (i.e., MCF-7 and MDA-MB-468 cells, respectively). The 242 and 445 protein hits identified with altered stabilities in these comparative analyses included a large fraction with no significant expression level changes. This suggests thermodynamic stability measurements create a new avenue for protein biomarker discovery. A number of the identified protein hits are known from other biochemical studies to play a role in tumorigenesis and cancer progression. This not only substantiates the biological significance of the protein hits identified using the SILAC-SPROX approach, but it also helps elucidate the molecular basis for their disregulation and/or disfunction in cancer.

Keywords: BT-474; MCF-7; MDA-MB-468; chemical denaturation; protein folding.

Figure 1

Schematic representation of the experimental workflow used in this work. Shown at the bottom are the protein unfolding curves extracted from the corresponding SILAC-SPROX behaviors shown for a wild type methionine-containing peptide.

Figure 2

Representative SILAC-SPROX data and associated protein unfolding curves obtained in this work. (A) Data obtained on the peptide AMEVDERPTEQYSDIGGLDK from 26S protease regulatory subunit 6A in the MCF-7 versus MDA-MB-468 cell line comparison. (B) Data obtained on the peptide AM(ox)EVDERPTEQYSDIGGLDK from 26S protease regulatory subunit 6A in the MCF-7 versus MDA-MB-468 cell line comparison. (C) Data obtained on the peptide DHASIQM(ox)NVAEVDKVTGR from 40S ribosomal protein S21 in the MCF-7 versus BT-474 cell line comparison. (D) Data obtained on the peptide NPEEAELEDTLNQVMVVFK from Cullin-1 in the MCF-7 versus BT-474 cell line comparison. (E) Data obtained on a subset of the non-methionine-containing peptides from 26S protease regulatory subunit 6A in the MCF-7 versus MDA-MB-468 cell line comparison including: DSYLILETLPTEYDSR (●), EKAPSIIFIDELDAIGTK (■), LKPGDLVGVNK (◆), QTYFLPVIGLVDAEK (▲), VDILDPALLR (▼). In (A) – (D), the solid lines represent the best fit of the data to Equations 1 or 2; the dashed curves represent the extracted SPROX curves; the vertical lines indicate the transition midpoints of the SPROX curves; ‘X’ indicates a data point that was not included in the regression analysis.

Figure 3

Evaluation of the expression level data generated in this work. (A) Global distribution of the differences between the expression level data generated in this work and those reported in reference 1 for the MCF-7 versus BT-474 (solid line) and MCF-7 versus MDA-MB-468 (dashed line) cell line comparisons. Expression level data reported in reference 1 were processed to generate ratios of protein abundance in the MCF-7 cell line versus the other cell line (BT-474 or MDA-MB-468). (B) and (C) Venn diagrams showing the overlaps between proteins with expression level changes > 2-fold identified in this work and those in reference 1 for the MCF-7 versus BT-474 (B) and MCF-7 versus MDA-MB-468 (C) cell line comparisons.

Figure 4

Protein classes observed in the proteins assayed and identified as hits with altered thermodynamic stabilities in the (A) BT-474 versus MCF-7 and (B) MDA-MB-468 versus MCF-7 cell line comparisons. Each protein class contained at least 4 proteins. Protein classes enriched in the hits are indicated with a “*”.