Mass spectrometry for sequencing protein binding epitopes in heparin/heparan sulfate

HS regulates a wide array of biological processes by binding and regulating the activity of proteins. The establishment that protein binding does not occur in a random fashion on the HS chains accentuated the need to develop new tools for structure-function relationship elucidation of heparinoids. MS has proved to be a powerful tool complementing the wide range of analytical techniques used to decipher the HS code. The current thesis aims to optimize mass spectral methods to study protein binding to heparin/HS oligosaccharides and elucidate structural features mediating this process. Chapter 2 describes a novel mass spectrometry compatible protocol combining size exclusion chromatography and hydrophobic trapping. The protocol aims to screen libraries of heparin oligosaccharides for those able to bind a protein of interest. In order to test the method for its ability to screen for specific binders, a model system for protein-heparin interaction was used, namely the interaction of ATIII with a heparin hexasaccharides. An LC/MS method previously used for chondroitin sulfate analysis was adopted to characterize the binders. The most abundantly recovered binders are the unacetylated compositions with 8 and 9 sulfates. The binders were specific in that they are biologically active as mea to inhibit FXa in vitro. Chapter 3 extends the previous method to screen HS octasaccharides libraries for ATIII binding since HS is the physiologically important heparinoid. Binders were subjected to CID MS2 and their fragmentation patterns were indicative of isomeric enrichment compared to the initial library. In chapter 3, we apply the screening protocol to the less characterized heparin-binding protein, FGF2 using an HS hexasaccharide library. An additional screening method consisting of studying the intact protein-oligosaccharide complex in the gas phase is used. Both methods show that non-reducing end-derived oligosaccharides bind with higher affinity to FGF2 forming a 1:1 FGF2:hexasaccharide complex. The ability of the high affinity binders to activate FGF2 signaling was determined using a cell proliferation assay. From the data, a two end model mechanism for FGF2 signaling on short NS domains was proposed Finally chapter 5 highlights the importance of the data acquired in chapters 2-4 and includes the author's future directions.