UBC Theses and Dissertations
Polymers for proteomics : development of polyglycidol-based soluble ester-linked aldehydes and their peptide binding characteristics Beaudette, Patrick Edmund
High molecular weight hyperbranched polyglycerol (HPG) was selected for development as a soluble polymer support for the targeted selection and release of primary amine containing peptides from a complex mixture. HPG has been functionalized with an ester-linked aldehyde group, that can be cleaved off under basic conditions, for the binding of primary amine containing peptides via a reductive alkylation reaction. Once bound, the high molecular weight of the polymer facilitates separation from a complex peptide mixture by employing either a 30-kDa molecular weight cut-off membrane or precipitation in acetonitrile. Following separation and washing of the polymer to remove unbound peptides and reagents, hydrolysis of the ester linker releases the bound peptide into solution for analysis by mass spectrometry. Released peptides retain the polymer linker moiety and are therefore characteristically mass-shifted. Four cleavable aldehyde polymers (PG1, PG2, PG3, and PG5) and a cleavable succinimidyl succinate polymer (PG4) have been prepared and characterized, each demonstrating the ability to selectively bind primary amine peptides from a complex mixture containing blocked (dimethylated tertiary amine) and unblocked (primary amine) peptides. The polymers have low non-specific binding properties and exhibit a range of reactivity and binding capacity. The specificity towards primary amines was designed to capture the cleavage products generated during proteolysis of a peptide library for the purpose of protease specificity profiling. PG2 was selected as the most promising candidate for having the best binding capacity of the tested polymers and no detectable non-specific binding effects.
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