Alterations in protein phosphorylation and glycosylation are an essential part of cell signaling mechanisms. Such changes in protein modification patterns may be directly involved in new pathways that lead to oncogenesis. It is thus essential to monitor changes in these patterns on a global scale and to be able to identify the proteins involved that may be critical in different stages of cancer progression. In order to monitor such changes on a global scale a top-down approach will be used. This strategy will involve a 2-D liquid based fractionation of intact proteins from tissue cell lysates. These fractionated proteins will be spotted onto protein microarrays for detection of phosphorylations based upon phospho-selective fluorescent dyes and antiphosphotyrosine antibodies and selective Ser/Thr phosphatases. Glycosylation patterns will also be mapped using fluorescent lectin probes on the protein microarrays. Changes in the presence of glycosylation and structural variations of glycans will be monitored using different types of lectins that are sensitive to glycan structure. Selected liquid fractionated proteins that correspond to spots that light up on the arrays will be directed into ESI-TOF MS to determine MW as a function of pi. Shifts in the pi value and the intact molecular weight of proteins identified by peptide mapping and MS/MS as compared to database values will be used together with the microarrays to map the phospho- and glyco-protein content of tumor samples. Sites of posttranslational modifications will be identified from protein digests using peptide microarrays, capillary monolithic LC-MALDI of intact proteins with on-plate digestion, RP-HPLC/ESI-LTQ MS/MS and MALDI QIT-TOF MS/MS. It will be shown that by using the total information provided by the intact MW value and ESI-MS and MALDI-TOF MS of the digests that very high sequence coverage can be obtained for any protein. An important part of this study will be to study such changes in PTMs in different stages and grades of prostate tissue and for a sufficient number of samples to search for markers that can be correlated with the progression of the disease. This research has important implications in public health in that it may provide a methodology for searching for markers of disease. These markers can be related to the stage of the disease and so may become important for personalized treatment, an important issue in clinical medicine.
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