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Probing orientation of immobilized humanized anti-lysozyme variable fragment by time-of-flight secondary-ion mass spectrometry.

ABSTRACT: As methods to orient proteins are conceived, techniques must also be developed that provide an accurate characterization of immobilized protein orientation. In this study, X-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of a surface immobilized variant of the humanized anti-lysozyme variable fragment (HuLys Fv, 26 kDa). This protein contained both a hexahistidine tag and a cysteine residue, introduced at opposite ends of the HuLys Fv, for immobilization onto nitrilotriacetic acid (NTA) and maleimide oligo(ethylene glycol) (MEG)-terminated substrates, respectively. The thiol group on the cysteine residue selectively binds to the MEG groups, while the his-tag selectively binds to the Ni-loaded NTA groups. XPS was used to monitor protein coverage on both surfaces by following the change in the nitrogen atomic %. SPR results showed a 10-fold difference in lysozyme binding between the two different HuLys Fv orientations. The ToF-SIMS data provided a clear differentiation between the two samples due to the intensity differences of secondary ions originating from asymmetrically located amino acids in HuLys Fv (histidine: 81, 82, and 110 m/z; phenylalanine: 120 and 131 m/z). An intensity ratio of the secondary ion peaks from the histidine and phenylalanine residues at either end of the protein was then calculated directly from the ToF-SIMS data. The 45% change in this ratio, observed between the NTA and MEG substrates with similar HuLys Fv surface coverages, indicates that the HuLys Fv fragment has opposite orientations on two different surfaces.

SUBMITTER: Baio JE

PROVIDER: S-EPMC3136626 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Probing orientation of immobilized humanized anti-lysozyme variable fragment by time-of-flight secondary-ion mass spectrometry.

Baio J E JE Cheng Fang F Ratner Daniel M DM Stayton Patrick S PS Castner David G DG

Journal of biomedical materials research. Part A 20110209 1

As methods to orient proteins are conceived, techniques must also be developed that provide an accurate characterization of immobilized protein orientation. In this study, X-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of a surface immobilized variant of the humanized anti-lysozyme variable fragment (HuLys Fv, 26 kDa). This protein contained both a hexahistidine tag and a cy ...[more]

PMID: 21308984

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Project description:To fully develop techniques that provide an accurate description of protein structure at a surface, we must start with a relatively simple model system before moving to increasingly complex systems. In this study, X-ray photoelectron spectroscopy (XPS), sum frequency generation spectroscopy (SFG), near-edge X-ray adsorption fine structure (NEXAFS) spectroscopy, and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of Protein G B1 (6 kDa) immobilized onto both amine (NH(3)(+)) and carboxyl (COO(-)) functionalized gold. Previously, we have shown that we could successfully control orientation of a similar Protein G fragment via a cysteine-maleimide bond. In this investigation, to induce opposite end-on orientations, a charge distribution was created within the Protein G B1 fragment by first substituting specific negatively charged amino acids with neutral amino acids and then immobilizing the protein onto two oppositely charged self-assembled monolayer (SAM) surfaces (NH(3)(+) and COO(-)). Protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by XPS. Spectral features within the SFG spectra, acquired for the protein adsorbed onto a NH(3)(+)-SAM surface, indicates that this electrostatic interaction does induce the protein to form an oriented monolayer on the SAM substrate. This corresponded to the polarization dependence of the spectral feature related to the NEXAFS N(1s)-to-?* transition of the ?-sheet peptide bonds within the protein layer. ToF-SIMS data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within Protein G B1 (methionine: 62 and 105 m/z; tyrosine: 107 and 137 m/z; leucine: 86 m/z). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary-ions stemming from the amino acid residues at either end of the protein. The 2-fold increase in this ratio, observed between the protein covered NH(3)(+) and COO(-) SAMs, indicates opposite orientations of the Protein G B1 fragment on the two different surfaces.

Project description:This study assesses the capability of high-resolution surface analytical tools to distinguish immobilized antibody orientations on patterned surfaces designed for antibody affinity capture. High-fidelity, side-by-side copatterning of protein A (antibody Fc domain affinity reagent) and fluorescein (antibody Fab domain hapten) was achieved photolithographically on commercial amine-reactive hydrogel polymer surfaces. This was verified from fluorescence imaging using fluorescently labeled protein A and intrinsic fluorescence from fluorescein. Subsequently, dye-labeled murine antifluorescein antibody (4-4-20) and antibody Fab and Fc fragments were immobilized from solution onto respective protein A- and fluorescein- copatterned or control surfaces using antibody-ligand affinity interactions. Fluorescence assays support specific immobilization to fluorescein hapten- and protein A-patterned regions through antigen-antibody recognition and natural protein A-Fc domain interactions, respectively. Affinity-based antibody immobilization on the two different copatterned surfaces generated side-by-side full antibody "heads-up" and "tails-up" oriented surface patterns. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis, sensitive to chemical information from the top 2 to 3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in "heads-up" regions, and ions from amino acids enriched in Fc domains for antibodies oriented in "tails-up" regions. Principal component analysis (PCA) improved the distinct TOF-SIMS amino acid compositional and ion-specific surface mapping sensitivity for each "heads-up" versus "tails-up" patterned region. Characteristic Fab and Fc fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domain- and structure-specific compositional differences by TOF-SIMS analysis. Since antibody immobilization and orientation are critical to many technologies, orientation characterization using TOF-SIMS could be very useful and convenient for immobilization quality control and understanding methods for improving the performance of antibody-based surface capture assays.

Project description:BackgroundTime-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive mass spectrometry technique with potential strengths as a method for detecting enzymatic activity on solid materials. In particular, ToF-SIMS has been applied to detect the enzymatic degradation of woody lignocellulose. Proof-of-principle experiments previously demonstrated the detection of both lignin-degrading and cellulose-degrading enzymes on solvent-extracted hardwood and softwood. However, these preliminary experiments suffered from low sample throughput and were restricted to samples which had been solvent-extracted in order to minimize the potential for mass interferences between low molecular weight extractive compounds and polymeric lignocellulose components.ResultsThe present work introduces a new, higher-throughput method for processing powdered wood samples for ToF-SIMS, meanwhile exploring likely sources of sample contamination. Multivariate analysis (MVA) including Principal Component Analysis (PCA) and Multivariate Curve Resolution (MCR) was regularly used to check for sample contamination as well as to detect extractives and enzyme activity. New data also demonstrates successful ToF-SIMS analysis of unextracted samples, placing an emphasis on identifying the low-mass secondary ion peaks related to extractives, revealing how extractives change previously established peak ratios used to describe enzyme activity, and elucidating peak intensity patterns for better detection of cellulase activity in the presence of extractives. The sensitivity of ToF-SIMS to a range of cellulase doses is also shown, along with preliminary experiments augmenting the cellulase cocktail with other proteins.ConclusionsThese new procedures increase the throughput of sample preparation for ToF-SIMS analysis of lignocellulose and expand the applications of the method to include unextracted lignocellulose. These are important steps towards the practical use of ToF-SIMS as a tool to screen for changes in plant composition, whether the transformation of the lignocellulose is achieved through enzyme application, plant mutagenesis, or other treatments.

Project description:The ability to orient active proteins on surfaces is a critical aspect of many medical technologies. An important related challenge is characterizing protein orientation in these surface films. This study uses a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum frequency generation (SFG) vibrational spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to characterize the orientation of surface-immobilized Protein G B1, a rigid 6 kDa domain that binds the Fc fragment of IgG. Two Protein G B1 variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimide-oligo(ethylene glycol)-functionalized gold and bare gold substrates. X-ray photoelectron spectroscopy was used to measure the amount of immobilized protein, and ToF-SIMS was used to measure the amino acid composition of the exposed surface of the protein films and to confirm covalent attachment of protein thiol to the substrate maleimide groups. SFG and NEXAFS were used to characterize the ordering and orientation of peptide or side chain bonds. On both substrates and for both cysteine positions, ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite to the cysteine surface position as compared with nonspecifically immobilized protein, indicating end-on protein orientations. Orientation on the maleimide substrate was enhanced by increasing pH (7.0-9.5) and salt concentration (0-1.5 M NaCl). SFG spectral peaks characteristic of ordered ?-helix and ?-sheet elements were observed for both variants but not for cysteine-free wild type protein on the maleimide surface. The phase of the ?-helix and ?-sheet peaks indicated a predominantly upright orientation for both variants, consistent with an end-on protein binding configuration. Polarization dependence of the NEXAFS signal from the N 1s to ?* transition of ?-sheet peptide bonds also indicated protein ordering, with an estimated tilt angle of inner ?-strands of 40-50° for both variants (one variant more tilted than the other), consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.

Dataset Information

Probing orientation of immobilized humanized anti-lysozyme variable fragment by time-of-flight secondary-ion mass spectrometry.

Publications

Probing orientation of immobilized humanized anti-lysozyme variable fragment by time-of-flight secondary-ion mass spectrometry.

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