To our knowledge, the current study is the first proteomic analysis of women with breast cancer using ICAT tandem MS and NAF. NAF was chosen as a substrate to evaluate biomarkers of breast disease because nipple aspiration is simple, quick, reliable, and reproducible [13–15, 35, 36]. In addition, nipple aspiration is completely noninvasive and requires a device with only minimal cost. Nipple aspiration provides concentrated secreted proteins not diluted with the irrigation fluid that is required to perform procedures such as ductoscopy or ductal lavage. Because of this, NAF is a rich source of proteins , thereby making it a good potential candidate as a source of biomarkers for early diagnosis of breast cancer [35, 37–40].
In the current study, ICAT identified 39 proteins that were differentially expressed in tumor-bearing versus disease-free breasts. Among the proteins identified, alpha2HS-glycoprotein was underexpressed, while lipophilin B, beta-globin, hemopexin, and vitamin D-binding protein precursor were overexpressed. A number of other proteins were identified, however these identifications had lower ProICAT scores. Despite being confirmed with manual curation, one may need to use Mascot or other secondary filters to eliminate the chance of false positive identification.
Alpha2HS-glycoprotein is important in blocking transforming growth factor-beta1 signal transduction, which is associated with tumor progression and resistance to chemotherapy in established cancers [41, 42]. Alpha2HS-glycoprotein has been observed to be depleted in certain tumors compared with normal tissue , consistent with the decreased ICAT ratio reported here.
Overexpression of lipophilin B, beta-globin, and hemopexin has also been confirmed in tumor-bearing tissues using non-ICAT techniques. Specifically, lipophilin B mRNA is overexpressed in 70% of breast tumors, and serum antibodies to lipophilin B have been detected in breast cancer patients [43–45]. Similarly, beta-globin has been found to be overexpressed in a number of different cancers compared with normal tissue [46–48]. Hemopexin, a protein involved in matrix metalloproteinase (MMPs) activation, has been linked to invasion and metastasis in many cancer model systems, including human breast cancer . MMP activation requires an intact hemopexin domain, and overexpression of hemopexin may increase MMP activity, degradation of extracellular matrix, and tumor cell migration [50, 51].
Our finding that vitamin D-binding protein precursor was overexpressed in the tumor-bearing breasts of women with breast cancer also agrees with previous findings. Receptors for 1,25-dihydroxyvitamin D have been shown to exist in cultured breast cancer cells and in primary breast cancers [32–34, 52]. In fact, the vitamin D-receptor has been reported to be present in 80% of breast cancers  and to be expressed at significantly higher concentrations in breast cancers than in normal breast tissue .
ICAT technology has been successfully used to study protein expression in yeast [16, 19], plant , and mammalian hematopoietic [25, 54] and liver cells . A review of the available literature reveals, however, that ICAT technology has not been employed in the clinical investigation of breast cancer. To date, only one study  has utilized stable isotope proteomic analysis to investigate ductal carcinoma of the breast. Zang et al.  compared protein expression between 10,000 cells each of microdissected normal ductal epithelium and metastatic ducal carcinoma using MS/MS in combination with16O/18O isotopic labeling introduced enzymatically. A total of 76 proteins were identified, and some of the proteins – such as mitochondrial isocitrate dehydrogenase, actin, and 14-3-3 protein xi/delta – were found to be significantly upregulated in breast tumor cells . Although similar to ICAT, the stable isotope technique used in these experiments did not employ an affinity component to enhance the separation of the labeled peptides.
ICAT technology is still in the early phase of development and should be regarded as a screening step to identify potentially interesting protein changes, which need to be independently confirmed by immunoblotting before they can be considered confirmed . In the current study, we performed western blot analysis on independent NAF samples to confirm the overexpression of vitamin D-binding protein that had been noted with ICAT. Due to limited sample material, immunoblotting could not be performed on the other proteins identified by ICAT. Vitamin D-binding protein was chosen because of its known association with breast cancer and because it had a high ICAT ratio (1.82), indicating significantly different expression in the tumor-bearing breast versus disease-free breasts. Western blot analysis confirmed that, in general, vitamin D-binding protein was significantly overexpressed in NAF obtained from women with breast cancer, although there was variation in expression among patients. Comparison of abundance ratios measured by ICAT and by Western blotting of the pooled specimens showed similar values (i.e., a two-fold increase in vitamin D-binding protein in the NAF from the tumor-bearing versus disease-free breasts). Previous reports have confirmed that the ICAT method is highly accurate for the quantification of proteins. The ICAT method has accurately predicted the relative quantification of proteins at known concentrations in mixtures [16, 19, 57, 58]. Comparison of abundance ratios obtained by ICAT and by Western blot analysis has also consistently shown similar values . In aggregate, these data strongly suggest that the ICAT method can be used to accurately measure the changes in abundance of differentially expressed proteins.
Reports in the literature indicate that ICAT technology can identify over 100 proteins per sample . The relatively small number of proteins identified in the current study was most likely related to the preponderance of a few highly abundant, but non-specific, proteins in NAF. Similar to serum and plasma, in which about 75% of the total protein is comprised of albumin or immunoglobulins, NAF has high levels of albumin, lactotransferrin, immunoglobulins, and apolipoprotein D. In the future, we intend to remove these proteins prior to ICAT analysis to facilitate the MS identification of other, potentially more relevant proteins. Newer stable-isotope technology such as the iTRAQ system (Applied Biosystems), which allows for the direct comparison of up to four samples in a single experiment rather than the two samples possible with ICAT, may be informative. Use of this strategy would allow the direct inclusion of two additional clinically relevant samples – such as NAF from women with ductal carcinoma in situ and NAF from women with fibrocystic disease.