Protein based microarrays: A tool for probing the proteome of cancer cellsand tissues

A novel proteomic approach for probing cell and tissue proteome, which comb ines liquid phase protein separations with microarray technology has been d eveloped. Proteins in cell and tissue lysates or in cellular subfractions a re separated using any one of a number of separation modes which may consis t of ion exchange liquid chromatography (LC), reverse phase LC, carrier amp holyte based separations, e.g. the use of Rotofor, affinity based separatio ns, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fra ctions with substantially reduced protein complexity. The advantage of a li quid based separation system is that proteins in hundreds of individual fra ctions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrom etry and may be further resolved to determine the nature of the reactive pr otein(s). We present in this report initial data based on microarray analys is of individual Rotofor fractions obtained from lung adenocarcinoma cell l ine A549 lysates which have been probed with antibodies against specific pr oteins.