Bioconjugation Techniques Research Articles

CrossSearch, a User-friendly Search Engine for Detecting Chemically Cross-linked Peptides in Conjugated Proteins

Chemical cross-linking and high resolution MS have been integrated successfully to capture protein interactions and provide low resolution structural data for proteins that are refractive to analyses by NMR or crystallography. Despite the versatility of these combined techniques, the array of products that is generated from the cross-linking and proteolytic digestion of proteins is immense and generally requires the use of labeling strategies and/or data base search algorithms to distinguish actual cross-linked peptides from the many side products of cross-linking. Most strategies reported to date have focused on the analysis of small cross-linked protein complexes (<60 kDa) because the number of potential forms of covalently modified peptides increases dramatically with the number of peptides generated from the digestion of such complexes. We report herein the development of a user-friendly search engine, CrossSearch, that provides the foundation for an overarching strategy to detect cross-linked peptides from the digests of large (>or=170-kDa) cross-linked proteins, i.e. conjugates. Our strategy combines the use of a low excess of cross-linker, data base searching, and Fourier transform ion cyclotron resonance MS to experimentally minimize and theoretically cull the side products of cross-linking. Using this strategy, the (alpha beta gamma delta)(4) phosphorylase kinase model complex was cross-linked to form with high specificity a 170-kDa betagamma conjugate in which we identified residues involved in the intramolecular cross-linking of the 125-kDa beta subunit between its regulatory N terminus and its C terminus. This finding provides an explanation for previously published homodimeric two-hybrid interactions of the beta subunit and suggests a dynamic structural role for the regulatory N terminus of that subunit. The results offer proof of concept for the CrossSearch strategy for analyzing conjugates and are the first to reveal a tertiary structural element of either homologous alpha or beta regulatory subunit of phosphorylase kinase.

Ultrasensitive Fluorescence Microscopy Studies of Protein Interactions with Functionalized Surfaces

Controlling protein adsorption to surfaces is an important issue in biotechnology, biomedicine and basic sciences. Proteins readily adsorb non-specifically to a variety of surfaces in a process known as biofouling, which is a major problem in the biomedical, environmental and industrial fields that can be solved by pre-treating the surfaces with adsorption-resistant materials. Additionally, surfaces can be structured and functionalized so as to immobilize proteins in specific patterns, orientations and/or densities, which is important in such areas as biosensors, biocatalysis, biotechnology, protein microarrays and single molecule biophysics. Here we review the various surface modifications and bioconjugation techniques that have been used in these areas. We also introduce novel fluorescence microscopy techniques, which have been employed to analyze the resistance of the surfaces to non-specific adsorption with detection limits down to the single molecule. A huge advantageous property of fluorescence microscopy is its sensitivity to the structure of individual, immobilized proteins by using Förster resonance energy transfer (FRET). Thus one can examine if the surface coating maintains the folded, functional form of specifically immobilized proteins. We then describe several important advances in studying slow timescale biophysical processes such as protein folding of single, specifically immobilized proteins. Future directions into which this field is expected to progress are also discussed.

Organosilane functionalization of InN surface

Direct coupling of aminosilane molecules onto the plasma hydroxylated InN surface is demonstrated. By using synchrotron-radiation photoelectron spectroscopy, the authors have studied the surface chemical properties of (3-aminopropyl)trimethoxysilane coupled InN. They have also found that the positively charged amine terminal groups on the functionalized InN surface can be utilized to bind negatively charged Au colloids (10nm diameter) in aqueous solution. Combining with the existing bioconjugation techniques for Au colloids, this approach opens up the possibility of nanoparticle-biomolecular hybrid systems for chemical and biosensing using electronic devices based on surface-functionalized InN.

Quantum dot bioconjugates for imaging, labelling and sensing

One of the fastest moving and most exciting interfaces of nanotechnology is the use of quantum dots (QDs) in biology. The unique optical properties of QDs make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations, in which traditional fluorescent labels based on organic molecules fall short of providing long-term stability and simultaneous detection of multiple signals. The ability to make QDs water soluble and target them to specific biomolecules has led to promising applications in cellular labelling, deep-tissue imaging, assay labelling and as efficient fluorescence resonance energy transfer donors. Despite recent progress, much work still needs to be done to achieve reproducible and robust surface functionalization and develop flexible bioconjugation techniques. In this review, we look at current methods for preparing QD bioconjugates as well as presenting an overview of applications. The potential of QDs in biology has just begun to be realized and new avenues will arise as our ability to manipulate these materials improves.

Gold nanolayer-encapsulated silica particles synthesized by surface seeding and shell growing method: near infrared responsive materials

Gold nanolayer-encapsulated silica particles whose optical resonance is located in 750–900 nm spectral region were synthesized by combining Sn (tin)-surface seeding and a shell growing process. The synthesized composite particles can be potentially used in wide biological fields, due to biocompatibility and a well-known bioconjugation technique of gold layer. Sn atoms, which can act not only as a catalytic surface for reduction of gold but also as a linker between silica surface and gold nanoparticles, were chemically deposited on hydroxylated silica particles. Then, we introduced another reductant with gold chloride in order to produce a multilayer of Au shell. In the process, Au shells grew by the reduction of additional gold ions on the Sn-functionalized silica surface and resulted in the subsequent coalescence and growth of the deposited gold nanoparticles. Finally, a complete gold nanoshell was formed on the silica surface by the one-step method, without a repeated coating process. The deposition of a gold nanolayer on the silica particles was easily controlled by the concentration ratio of Sn-functionalized silica particles and gold chloride solutions. Transmission electron microscopy (TEM) images and optical extinction spectra clearly showed that gold nanolayers were successfully deposited on the silica surface by the novel method. As the gold colloids attached on the silica surface grew, their optical plasmon peak became red-shifted until complete a gold shell was formed. After the gold shell was completed, the optical plasmon resonance became blue-shifted and the extinction spectra were functions of a relative ratio of the core to shell thickness.