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Introduction  The objective of this study was to identify cancer-associated protein expression patterns in bilateral matched nipple aspiration fluids using nanoscale reciprocal Cy3/Cy5 labeling and high-content antibody microarrays. This novel platform allows the pair-wise comparisons of the relative abundance of 512 different antigens using minimal NAF sample containing 1 µg of total protein.     Material and Methods  Matched NAF samples from two normal controls and 18 patients with early stage breast cancer (stage I/II, 13; DCIS, 2) or precancerous lesions (ADH, 3) were studied. Self-to-self and left-to-right comparisons of the normal controls were performed to determine antigen variations due to normal experimental and biological variability. Based on these two experiments, a stringency filter of 15% was applied to eliminate noise. Antigens were considered differentially expressed if there were a consistent >15% change on relative Cy3/Cy5 signals on reciprocal slides.     Results and Discussion  The number of differentially expressed antigens varied between 10 and 72 in tumor associated NAF samples, and no single antigen can be used as a “universal” marker to identify all patients. Antigens that are elevated in at least four patients were selected for further evaluations, including NME1, PTK2B, ARRB1, MRIP, GFRA1, APC, HSPD1, and SLP76. The validity of the antibody array findings was affirmed by single immunoassay on western blot; elevated expression of four of the selected markers in NAF is supported by published immunohistochemistry studies on breast cancer tissues.     Conclusions  Nipple aspiration fluid is a rich source of breast cancer biomarkers. This targeted proteomics approach for biomarker discovery is proven effective for clinical samples with limited protein content.

A Targeted Proteomics Approach for Biomarker Discovery Using Bilateral Matched Nipple Aspiration Fluids