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J Biomol Tech. 2010 September; 21(3 Suppl): S23.
PMCID: PMC2918118

Evaluating Protein Degradation Products as Serum Biomarkers to Determine the Invasiveness of Breast Carcinoma

J.A. Atwood, III,2 C. Shriver,3 B. Seth,4 D. Kirchner,4 R.J. Mural,4 and V.S. Kumar Kolli4
1Complex Carbohydrate Research Center (CCRC), University of Georgia, Athens, GA, United States;
2BioInquire, Athens, GA, United States;
3Walter Reed Army Medical Center, Washington, DC, United States;
4Windber Research Institute, Windber, PA, United States



The process by which cancer spreads from its site of origin to other tissues is a complex series of steps. The first of these involves the cancer cells freeing themselves from the primary tumor by degrading the proteins that make up the surrounding extracellular matrix (ECM), which separates the tumor from adjoining tissues. Degradation of the ECM is accomplished by the tumor cells secreting a variety of proteases, including the matrix metaloproteases (MMPs) and Plasminogen activators (PAs). Our hypothesis is that these secreted enzymes leak into the blood stream where they degrade serum proteins. Based on this assumption, we predict that the level of these fragments and their extent of degradation will be indicative of tumor invasiveness and thus capable of serving as serum biomarkers for this condition. Methods: The breast cancer serum (20 mL) samples (20 benign and 20 invasive, stageI) were processed with Enchant multi affinity protein separation kit to remove the high abundant proteins. Samples were separated on a 1D gel, the gel lanes were cut into segments and proteolytically digested, and the extracted peptides were run in duplicates on a LTQ-FT MS. MS/MS were searched against a database of human proteins with Sequest, and identifications were statistically validated at a 1% protein false discovery rate using the Provalt algorithm in ProteoIQ. Results/Conclusions: A GeLC-MS approach for this study because the intact proteins are separated via SDS gel electrophoresis. Hence, this sample-flow allows for the identification and relative quantification of proteins whose experimental MW (determined by SDS gel) differs significantly from their calculated MWs. This process has allowed us to identify a number of protein degradation products that appear to correlate well with the invasiveness of the tumors.

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