A novel phosphoprotein analysis scheme for assessing changes in premalignant and malignant breast cell lines using 2D liquid separations, protein microarrays and tandem mass spectrometry.

An analysis of phosphorylation changes that occur during cancer progression would provide insights into the molecular pathways responsible for a malignant phenotype. In this study we employed a novel coupling of 2D-liquid separations and protein microarray technology to reveal changes in phosphoprotein status between premalignant (AT1) and malignant (CA1a) cell lines derived from the human MCF10A breast cell lines. Intact proteins were first separated according to their isoelectric point and hydrophobicities, then arrayed on SuperAmine glass slides. Phosphoproteins were detected using the universal, inorganic phospho-sensor dye, ProQ Diamond. Using this dye, out of 140 spots that were positive for phosphorylation, a total of 85 differentially expressed spots were detected over a pH range of 7.2 to 4.0. Proteins were identified and their peptides sequenced by mass spectrometry. The strategy enabled the identification of 75 differentially expressed phosphoproteins, from which 51 phosphorylation sites in 27 unique proteins were confirmed. Interestingly, the majority of differentially expressed phosphorylated proteins observed were nuclear proteins. Three regulators of apoptosis, Bad, Bax and Acinus, were also differentially phosphorylated in the two cell lines. Further development of this strategy will facilitate an understanding of the mechanisms involved in malignancy progression and other disease-related phenotypes.