Quantification of particle-conjugated or particle-encapsulated peptides on interfering reagent backgrounds

Abstract

Particle-based technologies are increasingly being used in diagnostics and therapeutics. The particles employed in these applications are usually composed of polymers such as poly(lactide-co-glycolide) (PLG) and functionalized with peptides or proteins. Peptide or protein conjugation to particles is frequently achieved using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), while dimethyl sulfoxide (DMSO) is used to retrieve surface-attached or encapsulated peptides or proteins by solubilizing the particles. We examined strategies based on bicinchoninic acid (BSA), Coomassie Plus, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays for the quantification of surface-attached or encapsulated peptides or proteins. We determined that the CBQCA assay is a highly sensitive and accurate substitute for radioactivity-based assays that is suitable for measuring multiple particle-bound or particle-encapsulated peptides or proteins in the presence of EDC or PLG in DMSO, compounds that interfere with the more commonly used BSA and Coomassie Plus assays. Our strategy enables the accurate quantification of peptides or proteins loaded onto or into particles-an essential component of particle-based platform design for diagnostics and therapeutics.

Keywords: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde; bicinchoninic acid; microparticles; nanoparticles; peptides; poly(lactide-co-glycolide); proteins.

Figure 1. Interference of EDC or PLG in DMSO with the BCA or Coomassie Plus assays

(A) EDC interfered with the BCA assay and resulted in absorbance similar to BSA. (B) Absorption spectra of the purple-colored complexes produced by EDC (200 mg/mL) and BSA (250 μg/mL) in the BCA assay. (C) Kinetic studies of the reactions of EDC (200 mg/mL) and BSA (250 μg/mL) with the BCA reagent. (D) EDC did not interfere with the Coomassie Plus assay. (E) INS, LYS, OVA, BSA, PLP_139–151 and OVA_323–339, but not MBP, exhibited linear concentration-absorbance relationships with the Coomassie Plus assay. (F) PLG in DMSO interfered with the Coomassie Plus assay.

Figure 2. The CBQCA assay is suitable for use with multiple peptides and proteins and is not subject to interference from EDC or PLG in DMSO

(A) EDC did not interfere with the CBQCA assay. Fluorescence was measured in arbitrary units (a.u.). (B) PLG in DMSO did not interfere with the CBQCA assay. (C) INS, LYS, MBP, OVA, BSA, PLP_139–151 and OVA_323–339 exhibited linear concentration-fluorescence relationships with the CBQCA assay. (inset) Resolution and dynamic range of the CBQCA assay can be increased for OVA_323–339.