A targeted mass spectrometry immunoassay to quantify osteopontin in fresh-frozen breast tumors and adjacent normal breast tissues.

Osteopontin (OPN) is a multifunctional protein that can activate cell-signaling pathways and lead to cancer development and metastasis. Elevated OPN expression was reported in different cancer types, including breast tumors. Here, we present a new immuno-mass spectrometry method for OPN quantification in fresh-frozen malignant and adjacent normal human breast tissues. For quantification we used two proteotypic peptides: OPN-peptide-1 and OPN-peptide-2. Peptide concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode with stable isotope standards (SIS) and immuno-affinity enrichment for isolation of OPN peptides. Based on the OPN-peptide-1, the average OPN concentration in normal breast tissue was 19.42 μg/g, while the corresponding level in breast tumors was 603.9 μg/g. Based on OPN-peptide-2, the average concentration in normal breast tissue was 19.30 μg/g and in breast tumors 535.0 μg/g. In ER/PR/HER2(-) patients the OPN levels in breast tumors were significantly higher than in corresponding normal breast tissue samples, whereas in the single ER/PR/HER2(+) patient the OPN concentration in tumor samples was lower than in normal breast tissue sample. In conclusion, the current method is considered promising for the quantification of OPN in research and in clinical settings and should be further studied in breast cancer patients. SIGNIFICANCE: A new immuno-mass spectrometry method was successfully developed and applied to determine OPN concentrations in malignant tumor and normal breast tissues from six patients, and the method is promising for OPN quantification in both research and clinical settings.