Transferrin-bound proteins as potential biomarkers for advanced breast cancer patients

Abstract

Background: Serum profiling using mass spectrometry-based proteomic techniques has great potential to detect biomarkers that might improve the management for advanced breast cancer patients. The albuminome has previously been investigated as a tool in biomarker discovery, however other high abundant blood proteins are also likely to sequester potentially interesting molecules.

Methods: Affinity resin purified and isolated Transferrin and associated bound proteins from normal control and breast cancer patient serum samples were analysed by label-free mass spectrometry during the discovery phase.

Results: 21 significant proteins were identified with Fibrinogen and Fibronectin selected for further analysis in an independent sample set, with significant difference found when comparing the controls groups (normal healthy control, inflammatory bowel disease and benign breast disease) to stage IV breast cancer.

Conclusions: The area under the curve value for Fibrinogen compared favourably with cancer antigen 15-3, an established breast cancer tumour marker. A combination of all three biomarkers improved accuracy when comparing control/benign to stage IV breast cancer patient groups.

General significance: Mass spectrometry profiling of Transferrin-bound proteins has revealed serum proteins that can distinguish between serum from advanced breast cancer patients and healthy control subjects with high confidence.

Keywords: Biomarkers; Breast cancer; Fibrinogen; Fibronectin; Mass spectrometry; Transferrin.

Fig. 1

SDS-PAGE analysis of Transferrin-associated proteins. Samples were electrophoretically separated using a gradient (10–20%) polyacrylamide slab gel under reducing conditions with ß-mercaptoethanol. Aliquots of 20 μg were loaded and the gel was stained with Coomassie blue. Lane 1 — molecular weight standards; 3–12 Transferrin and Transferrin-associated proteins from normal healthy control and breast cancer patient serum samples. The most intense band at approximately 80 kDa was cut from the gel, digested and analysed by mass spectrometry. This band was identified as Transferrin (Mascot score 341).

Fig. 2

STRING analysis of 21 differentially expressed Transferrin-associated proteins listed in Table 2. The STRING programme generates functional protein association networks. This evidence view uses different-coloured lines to depict the type of evidence that supports each interaction. Highlighted in red are proteins involved in the complement and coagulation cascades with the majority of these found to have higher levels in breast cancer patient samples.

Fig. 3

Box and whisker plots (range, median, quartiles) of A. Fibronectin, B. Fibrinogen and C. CA15-3 in control, IBD (Inflammatory Bowel Disease), benign breast disease and stages I–IV breast cancer patient serum samples (validation sample set). Fibrinogen, Fibronectin and CA15-3 were all found to be statistically significant when comparing abundance levels between normal control and stage IV breast cancer patient serum samples. Both Fibrinogen and CA15-3 were also statistically significant comparing IBD or benign breast disease to stage IV breast cancer. Samples: normal healthy control (n = 54), inflammatory bowel disease (n = 19), benign breast disease (n = 12), stage I breast cancer (n = 20), stage II breast cancer (n = 20), stage III breast cancer (n = 39) and stage IV breast cancer (n = 76).