Hydrogel particles based on functionalized cross-linked poly-N-isopropylacrylamide co-polymers have proven to be powerful tools for the capture of low-molecular weight low-abundance peptides and proteins from biological samples. The harvesting characteristics of these particles reflect the physico-chemical properties of affinity baits incorporated in the particle and the properties of the polymer scaffold itself. To facilitate the identification of novel native CAMPs, we have engineered hydrogel particles that complement their physico-chemical properties, which tend to be low molecular weighted positively charged amphipathic peptides. Accordingly, we have focused on particles possessing both anionic and amphipathic characteristics that exclude larger proteins and peptides based on the cross-linking of the polymer scaffold. A combination of particles, with a distinct affinity bait formulations, have been successfully used to identify new CAMPs from plasma. In order to elevate the CAMP harvesting efficiency of the particles, we are now investigating how altering particle formulation and cross-linking impact their harvesting properties. We have also expanded the repertoire of monomers incorporated in the particles to investigate additional affinity baits to enhance selectivity and to increase particle stability so as to improve sample processing.
Lipid-Encapsulated Hydrogel Particles (Lipobeads)
Cationic antimicrobial peptides are generally believed to interact with bacterial membranes at some level. One strategy for preferentially enriching membrane-active peptides, such as cationic antimicrobial peptides, is to employ particles bearing lipid bilayers similar to those of bacterial membranes to harvest them. Accordingly, our lab is developing lipid-encapsulated hydrogel particles, lipobeads, for peptide harvesting applications. These particles consist of hydrophobically modified hydrogel particles encapsulated within an outer lipid bilayer. The outer membranes of these particles incorporate simplified lipid formulations designed to mimic those of bacteria. The hydrophobic modification of the core particle serves to anchor and stabilize the encapsulating membrane. In harvests from reptile plasma, particles with membrane properties similar to E. coli afforded dramatic preferential enrichment of peptides with physico-chemical characteristics associated with cationic antimicrobial peptides. We are currently investigating how lipid formulation impacts the harvesting properties of the particles, expanding to include formulations mimicking other bacteria such as P. aeruginosa, E. Faecilis and S. Aureus, as well as additional lipids such as cardiolipin, and phosphatidylethanolamine.
The CAMP harvest and capture project focus on specific species.
These species include but are not limited to:
- American Alligator (alligator mississipppiensis),
- Salt water crocodile (Crocodylus porosus),
- Komodo dragon (Varanus komodoensis)
Future species include:
- Boa Constrictors