Automated Solid-Phase Protein Modification with Integrated Enzymatic Digest for Reaction Validation: Application of a Compartmented Microfluidic Reactor for Rapid Optimization and Analysis of Protein Biotinylation

Abstract

Protein modification by covalent coupling of small ligands or markers is an important prerequisite for the use of proteins in many applications. Well-known examples are the use of proteins with fluorescent markers in many in vivo experiments or the binding of biotinylated antibodies via biotin-streptavidin coupling in the frame of numerous bioassays. Multiple protocols were established for the coupling of the respective molecules, e.g., via the C and N-terminus, or via cysteines and lysines exposed at the protein surface. Still, in most cases the conditions of these standard protocols are only an initial guess. Optimization of the coupling parameters like reagent concentrations, pH, or temperature may strongly increase coupling yield and the biological activity of the modified protein. In order to facilitate the process of optimizing coupling conditions, a method was developed which uses a compartmented microfluidic reactor for the rapid screening of different coupling conditions. In addition, the system allows for the integration of an enzymatic digest of the modified protein directly after modification. In combination with a subsequent MALDI-TOF analysis of the resulting fragments, this gives a fast and detailed picture not only of the number and extent of the generated modifications but also of their position within the protein sequence. The described process was demonstrated for biotinylation of green fluorescent protein. Different biotin-excesses and different pH-values were tested in order to elucidate the influence on the modification extent and pattern. In addition, the results of solid-phase based modifications within the microfluidic reactor were compared to modification patterns resulting from coupling trials with unbound protein. As expected, modification patterns of immobilized proteins showed clear differences to the ones of dissolved proteins.

Keywords: biotinylation; enzymatic digest; microfluidic reactor; protein modification; solid-phase reaction.

Figure 1

Chemical structure of N-hydroxysuccinimide (NHS)–biotin.

Figure 2

Scheme for analysis of the biotinylated modification sites.

Figure 3

Setup of the compartmented reactor system. (A) Scheme showing the different areas. (B) Photo showing the actual system.

Figure 4

(A) Titration curves of the DEAP, C22 particles, and water as control. 20 mg particles in 10 mL of a solution of 0.01 M NaOH were titrated with 0.1 M HCl. (B) For determination of an additional pKa of the DEAP particles, a difference plot of the DEAP and water titration curve was created and derived.

Figure 5

Mass spectra after biotinylation of eGFP at different pH-values. A 20-fold excess of biotin compared to the used 0.3 mg protein was applied.