The traditional method of proteomic analysis is one- or two-dimensional polyacrylamide gel electrophoresis (2-D-PAGE). Using two-dimensional rather than one-dimensional PAGE allows better separation of proteins of equal molecular weight based on charge. Once a protein of interest is found, it can be cut from the gel and identified. 2-D-PAGE has been used to screen NAF because it provides a convenient and rapid method for protein identification based on matrix-assisted laser desorption-time-of-flight mass spectrometry (MALDI-TOF MS). At least two studies have analyzed the NAF proteome. One (Varnum et al., 2003) used liquid chromatography, while the second used 2-D-PAGE (Alexander et al., 2004). More than 60 proteins were identified in the first and 41 in the second study. Many of the proteins were the same, but a significant subset of proteins (35 in the first, 21 in the second) were unique to each study. Both studies should be considered when assessing the NAF proteome.
2-D-PAGE may serve as a screening platform to identify proteins in NAF that are differentially expressed in cancerous and benign breasts. These proteins can then be validated using one or more high-throughput proteomic approaches (Alexander et al., 2004). Three protein spots were detected using 2-D-PAGE that were upregulated in three or more NAF samples from breasts with cancer. These spots were identified to be gross cystic disease fluid protein (GCDFP)-15, apolipoprotein (apo)D, and alpha-1 acid glycoprotein (AAG). To validate these three potential biomarkers, 105 samples (53 from benign breasts and 52 from breasts with cancer)
were analyzed using enzyme-linked immunosorbent assay (ELISA), a high-throughput method of evaluating protein concentration. Considering all subjects, GCDFP-15 levels were significantly lower and AAG levels significantly higher in breasts with cancer. This was also true in pre- but not postmenopausal women. GCDFP-15 levels were lowest and AAG levels highest in women with DCIS. Menopausal status influenced GCDFP-15 and AAG more in women without than with breast cancer. ApoD levels did not correlate significantly with breast cancer.
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