4T1 tumor cells were lysed and the cell extract was then mixed with biotin-labeled macroketone or free biotin for 2 hours at 4°C with gentle rotation. Recombinant streptavidin-agarose beads (Pierce, IL) were added to the cell extract and the incubation was continued for another 2 hours with gentle rotation. The mixtures were then loaded onto the Poly-Prep chromatography column. After extensive wash with cell lysis buffer, samples were eluted by elution buffer. The elute was further concentrated by Centricon P-20 (Millipore, MA) and separated by SDS-PAGE. The gel was stained by Coomassie Blue and the indicated band was cut out for mass spectrometry and peptide sequencing analyses.