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

Abstract

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.

Figure 1

Schema of ICAT analysis performed using a Cleavable ICAT Reagent Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's guidelines, with some modifications. Avidin Affinity refers to the Avidin Affinity Cartridge kit (Applied Biosystems, Foster City, CA)

Figure 2

Using the ProICAT bioinformatics program, 353 peptides were identified. Approximately equal numbers of proteins were up- versus down-regulated.

Figure 3

Quantification of vitamin D-binding protein through the ICAT-labeled parent ion signals of the peptide VC*SQYAAYGEK.

Figure 4

Identification of the peptide VC*SQYAAYGEK from vitamin D-binding protein by MS/MS.

Figure 5

Western blot analysis of independent NAF sample set. The relative difference in vitamin D-binding protein expression in the pooled samples of NAF from healthy volunteers versus NAF from women with early-stage breast cancer was two-fold (P = 0.04; standard error = 9703.7 (solid bars). Individual NAF specimens showed variation in vitamin D-binding protein expression among both healthy volunteers and women with early-stage breast cancer (dotted bars). Menopausal status did not affect expression of vitamin D-binding protein in NAF.