Jos Buijs

Protein adsorption on solid surfaces

The research field of protein adsorption on surfaces appears to be as popular as ever. In the past year, several hundred published papers tackled problems ranging from fundamental aspects of protein surface interactions to applied problems of surface blood compatibility and protein surface immobilization. Although some parts of the protein adsorption process, such as kinetics and equilibrium interactions, can be accurately predicted, other aspects, such as the extent and the rate of protein conformational change, are still somewhat uncertain. The whole field is ripe for a comprehensive theory on protein adsorption.

Methods for Studying Protein Adsorption

Proteins are interfacially active molecules; a statement that is demonstrated easily by the spontaneous accumulation of proteins at interfaces.1–4 Why do proteins show the propensity to adsorb to interfaces and why do they adsorb so tenaciously? For some proteins, the tendency to adsorb is due to the nature of side chains present on the surface of the protein. Protein is an amphoteric polyelectrolyte.5 Its amino acids have different characteristics: some are apolar and like to be buried inside the protein globule, whereas others are polar and charged and are often found on the outside protein surface. A strong, long-ranged electrostatic attraction between a charged adsorbent and oppositely charged amino acid side chains will lead to a significant free energy change favoring the adsorption process. In other cases, the interfacial activity of the protein may be driven by its marginal structural stability.6 The compactness of the native structure of the protein is due to the optimal amount of apolar amino acid residues. The stability of such a structure depends on the combination of hydrophobic interactions between the hydrophobic side chains, hydrogen bonds between the neighboring side chains and along the polypeptide chains, and the Coulomb interactions between charged residues and van der Waals interactions. An adsorbent surface can “compete” for the same interactions and minimize the total free energy of the system by unfolding the protein structure: the adsorption process may result in a surface-induced protein denaturation.7,8 Elements of the secondary structure of the protein (α helix and β sheet) together with the supersecondary motifs form a compact globular domain. Some proteins are built from more than one domain. In a multidomain protein, it is possible that one domain will dominate the adsorption property of the whole macromolecule at a particular type of interface. For example, acid-pretreated antibodies bind with their constant fragments to a hydrophobic surface.9 In order to completely characterize and predict protein adsorption, one would like to have a quantitative description of adsorption. This description is typically obtained by measuring the adsorption isotherm, adsorption and desorption kinetics, conformation of adsorbed proteins, number and character of protein segments in contact with the surface, and other physical parameters related to the adsorbed protein layer, such as layer thickness and refractive index. This article describes a selected set of techniques and protocols that will provide answers about the mechanism of protein adsorption onto and desorption from surfaces. The reader is referred to the specialized monographs1–4 and a review 10 on protein adsorption for a more comprehensive coverage of various aspects of protein–surface interactions.

Automatic analysis of hydrogen/deuterium exchange mass spectra of peptides and proteins using calculations of isotopic distributions.

High mass-resolving power has been shown to be useful for studying the conformational dynamics of proteins by hydrogen/deuterium (H/D) exchange. A computer algorithm was developed that automatically identifies peptides and their extent of deuterium incorporation from H/D exchange mass spectra of enzymatic digests or fragment ions produced by collisionally induced dissociation (CID) or electron capture dissociation (ECD). The computer algorithm compares measured and calculated isotopic distributions and uses a fast calculation of isotopic distributions using the fast Fourier transform (FFT). The algorithm facilitates rapid and automated analysis of H/D exchange mass spectra suitable for high-throughput approaches to the study of peptide and protein structures. The algorithm also makes the identification independent on comparisons with undeuterated control samples. The applicability of the algorithm was demonstrated on simulated isotopic distributions as well as on experimental data, such as Fourier transform ion cyclotron resonance (FTICR) mass spectra of myoglobin peptic digests, and CID and ECD spectra of substance P.

The effect of adsorption on the antigen binding by IgG and its F(ab′)2 fragments

Abstract The adsorption process of two monoclonal IgGs and their F(ab′)2 fragments is related to the efficiency with which these adsorbed proteins bind antigens. Adsorption and antigen binding experiments are performed under various conditions of hydrophobicity of the sorbent surface, pH and ionic strength. The immobilization of proteins is followed in time using the optical technique reflectometry. From previous results it has been inferred that the Fc part of an IgG molecule is structurally less stable than the F(ab′)2 part. This lower structural stability promotes adsorption of the IgG molecules with their Fc parts to the sorbent surface, directing the Fab parts towards the solution. Indeed, it is found that the antigen binding ratios are higher under adsorption conditions at which the affinity of F(ab′)2 fragments for the sorbent surface is relatively low. Furthermore, it is observed that the orientation of an adsorbed IgG molecule with an uneven charge distribution can be strongly influenced by electrostatic interactions. This results in a total absence of immunological activity when the Fab parts are strongly electrostatically attracted by the sorbent surface.

Localized changes in the structural stability of myoglobin upon adsorption onto silica particles, as studied with hydrogen/deuterium exchange mass spectrometry

A new method is presented for monitoring the conformational stability of various parts of a protein that is physically adsorbed onto nanometer-sized silica particles. The method employs hydrogen/deuterium (H/D) exchange of amide hydrogens, a process that is extremely sensitive to structural features of proteins. The resulting mass increase is analyzed with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Higher structural specificity is obtained by enzymatically cleaving the adsorbed proteins prior to mass spectrometric analysis. The mass increases of four peptic fragments of myoglobin are followed as a function of the H/D exchange time. The four peptic fragments cover 90% of the myoglobin structure. Two of the peptic fragments, located in the middle of the myoglobin sequence and close to the heme group, do not show any adsorption-induced changes in their structural stability, whereas the more stable C- and N-terminal fragments are destabilized. Interestingly, for the N-terminal fragment, comprising residues 1-29, two distinct and equally large conformational populations are observed. One of these populations has a stability similar to that in solution (-23 kJ/mol), whereas the other population is highly destabilized upon adsorption (-11 kJ/mol).

Tobacco mosaic virus adsorption on self-assembled and Langmuir–Blodgett monolayers studied by TIRF and SFM

The adsorption of tobacco mosaic virus (TMV) on self-assembled and Langmuir-Blodgett monolayers was investigated using total internal reflection fluorescence (TIRF) spectroscopy and scanning force microscopy (SFM). Substrates were chosen to probe electrostatic, hydrophobic and surface fluidity effects on TMV adsorption. Positively charged and hydrophobic surfaces demonstrated similar initial rates of TMV adsorption; however, their respective surface TMV coverages differed greatly. Likewise, positively charged surfaces which differed primarily in surface fluidity exhibited similar adsorption rates for TMV, but different TMV surface coverages. In contrast, virus adsorption to negatively charged and zwitterionic substrates was negligible. To elucidate these differences in adsorption behavior, SFM was used to image the distribution and the aggregation state of adsorbed TMV.

Inter- and intra-molecular migration of peptide amide hydrogens during electrospray ionization

The isotopic exchange of amide hydrogens in proteins in solution strongly depends on the surrounding protein structure, thereby allowing structural studies of proteins by mass spectrometry. However, during electrospray ionization (ESI), gas phase processes may scramble or deplete the isotopic information. These processes have been investigated by on-line monitoring of the exchange of labile deuterium atoms in homopeptides with hydrogens from a solvent suitable for ESI. The relative contribution of intra- and inter-molecular exchange in the gas phase could be studied from their distinct influence on the well-characterized exchange processes in the spraying solution. The deuterium content of individual labile hydrogens was assessed from the isotopic patterns of two consecutive collision-induced dissociation fragments, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Results demonstrate that gas phase exchange in the high-pressure region between the capillary and the skimmer cause substantial depletion of the isotopic information of penta-phenylalanine and penta-aspartic acid. For penta-alanine and hexa-tyrosine, the amide hydrogens located close to the N-terminus are depleted from deuterium during mass analysis. Amide hydrogens located close to the C-terminus still retain the information of the isotopic state in solution, but they are redistributed by intra-molecular exchange of the amide hydrogens with the C-terminal hydroxyl group.

A new method for the accurate determination of the isotopic state of single amide hydrogens within peptides using Fourier transform ion cyclotron resonance mass spectrometry

A new method is presented to accurately determine the probability of having a deuterium or hydrogen atom on a specific amide position within a peptide after deuterium/hydrogen (D/H) exchange in solution. Amide hydrogen exchange has been proven to be a sensitive probe for studying protein structures and structural dynamics. At the same time, mass spectrometry in combination with physical fragmentation methods is commonly used to sequence proteins based on an amino acid residue specific mass analysis. In the present study it is demonstrated that the isotopic patterns of a series of peptide fragment ions obtained with capillary-skimmer dissociation, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, can be used to calculate the isotopic state of specific amide hydrogens. This calculation is based on the experimentally observed isotopic patterns of two consecutive fragments and on the isotopic binomial distributions of the atoms in the residue constituting the difference between these two consecutive fragments. The applicability of the method is demonstrated by following the sequence-specific D/H exchange rate in solution of single amide hydrogens within some peptides.

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111In-labelled DOTA-conjugated ZEGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-ZEGFR:2377. DOTA-ZEGFR:2377 was labelled with 57Co (T1/2 = 271.8 d), 55Co (T1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68Ga (T1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57Co was used in animal studies. Both 57Co-DOTA-ZEGFR:2377 and 68Ga-DOTA-ZEGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-ZEGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57Co-DOTA-ZEGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68Ga-DOTA-ZEGFR:2377. The results of this study suggest that the positron-emitting cobalt isotope 55Co would be an optimal label for DOTA-ZEGFR:2377 and further development should concentrate on this radionuclide as a label.

ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers.

Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, ¹¹¹In for SPECT imaging and ⁶⁸Ga for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule (111)In/⁶⁸Ga-DOTA-(HE)3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.