Protein identification from dried nipple aspirate fluid on Guthrie cards using mass spectrometry

Nipple aspirate fluid (NAF) requires investigation as a potential source of biomarkers for early diagnosis or risk assessment in breast cancer and other breast disorders. The present study demonstrated that proteins were easily extracted from dried NAF spots on Guthrie cards and were suitable for mass spectrometry analysis. NAF was obtained from 80 women, collected on Guthrie cards, between 2007 and 2010. The NAF-proteins were extracted from the card by incubating the card in water. These proteins were then quantified and separated using one-dimensional, 12% SDS-PAGE, gel electrophoresis and on high-resolution gradient gels at different concentrations (4-12, 8-16 and 4-20%). The bands with the most abundant proteins were excised from the gradient gels and the proteins were identified by liquid chromatography quadrupole time of flight. Immunoglobulins, Zn-α2-glicoprotein, apoliprotein D and prolactin inducible protein were among those identified. The NAF-Guthrie card collection method has not been applied previously, however, NAF proteins have been identified using other collecting techniques, confirming the feasibility of the NAF Guthrie card collection method for analyzing the proteins within NAF. The NAF-Guthrie card collecting method has multiple advantages, including being inexpensive, non-invasive, reliable and painless, and the cards can be stored at room temperature. Examining NAF may assist in identifying individuals at a higher risk of breast cancer and in improving patient prognosis.