Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

Background: Isotope-coded affinity tag ( ICAT) tandem mass spectrometry ( MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. Methods: Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women ( 8 with early-stage breast cancer and 4 healthy volunteers) was also performed. Results: 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up-versus down-regulated. Alpha2HS-glycoprotein [ Heavy: Light ( H: L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B ( H: L ratio 1.42), beta-globin ( H: L ratio 1.98), hemopexin ( H: L ratio 1.73), and vitamin D-binding protein precursor ( H: L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. Conclusion: ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.