Oriented Peptide Immobilization on Microspheres

Abstract

Reproducible immobilization of peptides and proteins on microsphere surfaces is a critical factor for optimal sensitivity and selectivity in bead-based assays. However, peptides with unusually large numbers of lysine residues-whose amines are targeted in the most common microsphere immobilization chemistries-may be particularly challenging to use in bead-based arrays, as they may lose activity through multipoint attachments and incorrect presentation. For this reason, it is imperative to achieve site-directed attachment chemistry, such that a single site of attachment provides reproducibly oriented peptides on the microsphere surface. This can be achieved by inserting a unique targetable residue, such as a cysteine. Here, we present methods for attaching cysteine-containing peptides to standard carboxy-functionalized microsphere surfaces using thiol- rather than amine-directed chemistries. We show that the presence of a cationic detergent (CTAB) and a "passivating" agent such as β-mercaptoethanol facilitates improved bead recovery after peptide immobilization and may enhance functionality of the attached peptides.

Effect of Linker Length on Cell Capture by Poly(ethylene glycol)-Immobilized Antimicrobial Peptides

“…Carboxy-functionalized MagPlex beads (25 μL; Luminex Corporation, Austin, TX, USA) were functionalized with antimicrobial peptides using linkers with various length maleimide-PEG-amine linkers as previously described. 29 Briefly, carboxyl beads were first treated with EDC and sulfo-NHS (5 mg/mL each) for 30 min at pH 6, rinsed, and then incubated with 1 mg/mL maleimide-PEG n -amine linker (pH 6) for 2 h. After additional rinsing, beads were mixed with peptides (0.25 mg/mL in 0.1 M tricine, pH 7.4, 0.05% CTAB), incubated overnight, and subjected to a final rinse to remove nonimmobilized peptides. To estimate the relative density of attached peptides, peptide-functionalized beads were treated for 60 min with biotin-LC-LC-NHS at 1.9, 7.5, or 30 mg/mL in PBS, pH 7.3.…”