Mass spectrometry (LC-MS/MS) analysis of proximal fluid and cell membrane proteins for breast cancer biomarkers

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 4436 With the advances in mass spectrometry, protein biomarker research represents a promising approach in disease detection and treatment. Protein profiling of tumor cells is the most direct way to study cancer signatures. However, plasma/serum is the most suitable clinical specimen for biomarker research because it is attainable by non-invasive means, and it is likely to contain tumor markers albeit in great dilution. However, low abundance and the interference of 20 high abundant proteins that make up 99% of serum proteins, complicates serum analyses. Therefore, it is necessary to have suitable starting material, enrichment and subfractionation methods, and a sensitive highly accurate procedure. Since many membrane proteins are released into the plasma/serum by exterior protein cleavage, membrane sloughing, and cell lysis, they represent promising candidates for interrogation. Because such proteins are frequently glycosylated, we chose the hydrazide method to specifically target, enrich, and identify glycosylated proteins using the LTQ Orbitrap mass spectrometer. Our initial goal was to identify membrane proteins in breast cancer cell lines and the proteins secreted into the proximal fluid, and then to test the hydrazide method with both media as a prelude to working with the highly diluted plasma/serum. The proteins are denatured and trypsinized, then the sugar residues are oxidized before reacting with the hydrazide resin. The covalently attached glycopeptides are then thoroughly washed of contaminants and PNGase F is then used to enzymatically release the N-linked peptides leaving signature deaminated asparagine residues modified to an aspartic acid. The samples are then processed by data dependant LC-MS/MS. The combination of steps facilitates identification of the glycopeptide and also the glycosylation site. In an initial MS screening over 200 proteins were found secreted into the proximal fluid of each breast cancer cell line, MCF-7 and 453, with over 1000 peptides identified in each case. Interestingly, two potential candidate biomarkers, cathepsin D and the galectin 3-binding protein were detected in the proximal fluid without the hydrazide method, and identified in breast cancer cell line membrane fractions using the hydrazide method. In turn, the N-linked glycosylation on the peptides are site mapped. Analyzing the membranes and proximal fluid of breast cancer cell lines has demonstrated the usefulness of the procedure for generating a large set of potential biomarkers. The method is capable of enriching the samples and allowing for the site mapping of glycopeptides in complex mixtures. The detection of cathepsin D and galectin 3-binding protein in the proximal fluid and the ability of the hydrazide method to enrich for these glycopeptides in complex membrane fractions appears to be a promising application for the enrichment of candidate biomarkers in the plasma/serum.