X-ray Photoelectron Spectroscopy (XPS): Principles, Data Interpretation, and Applications

X-ray photoelectron spectroscopy (XPS), also known as electron spectroscopy for chemical analysis (ESCA), is a powerful surface sensitive analytical technique. It's primarily used to study the elemental composition, chemical state, and electronic state of materials. In structural biology, XPS plays a critical role in the study of the interactions and properties of biomolecules at surfaces and interfaces. Although not traditionally associated with structural biology, its ability to probe molecular environments, such as protein-ligand interactions or surface modification of biomaterials, has opened new possibilities for understanding complex biological systems.

Creative Biostructure offers a comprehensive resource on X-ray photoelectron spectroscopy. As a leading structural biology services provider, we specialize in X-ray crystallography, cryo-electron microscopy (Cryo-EM), NMR spectroscopy, and more to support your protein structure research.

Introduction to X-rays and Their Role in Structural Biology

X-rays are a type of high-energy electromagnetic radiation with wavelengths ranging from 0.01 to 10 nanometers (nm), located between ultraviolet light and gamma rays on the electromagnetic spectrum. Discovered by Wilhelm Conrad Röntgen in 1895, X-rays are known for their ability to penetrate matter and interact with electrons, enabling applications ranging from medical imaging to materials science. In structural biology, X-rays are essential for elucidating the atomic-level architecture of biological macromolecules such as proteins, nucleic acids, and viruses.

X-ray Based Structural Biology Techniques

Impact of X-ray Techniques on Structural Biology

X-ray-based techniques, particularly X-ray crystallography, have revolutionized structural biology by providing atomic-level insight into biomolecular structures. These advances have made significant contributions to drug discovery, virology, and the elucidation of enzyme mechanisms, enabling researchers to design targeted therapies and understand fundamental biological processes.

Drug Discovery: Advancing Structure-Based Drug Design (SBDD)

X-ray crystallography is a cornerstone of structure-based drug design (SBDD), allowing researchers to visualize at high resolution how drug candidates interact with their molecular targets. By understanding these interactions, scientists can optimize drug binding, increase specificity, and improve efficacy, accelerating the development of new therapeutics.

A landmark example is Gleevec (Imatinib), a breakthrough treatment for chronic myeloid leukemia (CML). X-ray crystallography revealed how Gleevec specifically binds to BCR-ABL kinase, a mutated enzyme that drives leukemia progression. This insight enabled the rational design of targeted inhibitors, leading to a paradigm shift in cancer treatment.

Virology: Accelerating Vaccine and Antiviral Development

X-ray techniques have been instrumental in deciphering viral protein structures, providing critical information for the design of vaccines and antiviral drugs. Rapid structural resolution of the SARS-CoV-2 spike protein was critical to the development of mRNA vaccines and monoclonal antibody therapies during the COVID-19 pandemic.

In addition to SARS-CoV-2, X-ray crystallography has aided the understanding of other viral proteins, such as influenza hemagglutinin (HA), HIV reverse transcriptase, and Ebola virus glycoproteins, contributing to vaccine design and antiviral drug discovery.

Enzyme Mechanisms: Unraveling Catalytic Functions

X-ray crystallography has provided detailed structural insights into enzyme function and catalysis, revealing the precise binding sites, conformational changes, and reaction mechanisms that drive biochemical reactions.

Key discoveries include:

• ATP Synthase: X-ray studies revealed how ATP synthase couples proton gradients to ATP synthesis, elucidating the molecular rotation mechanism essential for cellular energy production.
• DNA Polymerases: Structural studies of DNA polymerases have clarified their role in DNA replication, repair, and proofreading, enabling the development of polymerase inhibitors as antiviral and anticancer agents.

Principle of XPS

The basic principle of XPS is simple and elegant. When a material is exposed to X-rays, electrons are ejected from the inner orbitals of the atoms. These emitted electrons are then detected and their energy measured. The energy of the emitted electron depends on the binding energy of the electron in its original atomic orbital. By analyzing these energies, one can deduce the elemental composition and chemical state of the atoms at the surface. The depth of analysis is limited to the top few nanometers of the material, making XPS ideal for surface analysis.

Figure 1. XPS physics - the photoelectric effect.

XPS Technique and Procedure

The process begins by irradiating the sample with X-rays. The X-rays cause electrons to be ejected from the atoms of the sample. These electrons are then collected by an electron analyzer, which measures their kinetic energy. The binding energy of the ejected electrons can be calculated by subtracting the measured kinetic energy from the energy of the incoming X-rays. This data is used to identify the elements present on the surface of the sample, as well as their chemical states.

A notable feature of XPS is its ability to provide information about chemical bonding. For example, the spectrum of an element may show multiple peaks corresponding to different oxidation states or different chemical environments, providing insight into chemical interactions at the surface. A typical XPS spectrum will show peaks corresponding to nuclear level electron binding energies, and the shifts in these peaks can be used to infer details about the bonding environment of the element.

Figure 2. Basic components of a monochromatic XPS system.

XPS Spectrum

An XPS spectrum typically shows the number of electrons detected at specific binding energies. Each element produces a distinct set of characteristic peaks corresponding to the electron configurations of its atoms, such as 1s, 2s, 2p, 3s, and so on. The intensity of each peak is proportional to the amount of that element in the sample volume. To calculate atomic percentages, the raw XPS signals are adjusted by dividing the peak intensities by relative sensitivity factors (RSFs) and normalizing the values over all detected elements. Note that hydrogen is not detected by XPS, so these atomic percentages exclude hydrogen.

Interpreting the XPS Spectrum

The XPS spectrum is analyzed by identifying the positions of the peaks, their intensities, and the shape of the spectra. The following steps are typically involved in the interpretation:

• Identification of Elements: The positions of the peaks are compared to known binding energies for different elements.
• Chemical State Analysis: Shifts in peak positions indicate changes in the chemical state of the elements. For example, the presence of a peak with a higher binding energy may indicate oxidation, while a lower binding energy may indicate reduction.
• Quantitative Analysis: The relative intensities of the peaks can be used to calculate the elemental composition, although factors such as cross sections and detection efficiency must be taken into account.

Figure 3. XPS spectra of the samples. a Broad scan of the sample, and b spectra of the Bi 4f electronic binding energy. c Fe 2p electronic binding energy spectra. d Oxygen 1s spectrum of BiFeO3 deposited with O:Ar = 1:0. e Oxygen 1s spectrum of BiFeO3 deposited with an O:Ar ratio 1:1. f Oxygen 1s spectrum of BiFeO3 deposited with an O:Ar ratio of 0:1. The peak at 530.7 eV corresponds to oxygen vacancies, and the peak at 532.6 eV corresponds to the adsorbed oxygen. The relative intensity ratios (RIR) of the oxygen vacancies to adsorbed oxygen were 0.101 (for 0% oxygen and 100% argon), 0.111 (for 50% oxygen and 50% argon), and 0.145 (for 100% oxygen and 0% argon), respectively. (Dayal and Jinesh, 2023)

Applications of XPS in Structural Biology

Protein-Surface Interactions

Proteins and other biomolecules often interact with surfaces, whether they are part of cellular membranes, scaffolds in tissue engineering, or sensors in diagnostic devices. XPS can be used to study how proteins adsorb to surfaces, how their conformation changes, or even how surface chemistry affects their activity. By analyzing the elemental composition of the surface and the binding states of proteins, researchers can gain a better understanding of the biomolecular interactions at play.

For example, in the study of biomaterials, XPS can provide insight into how proteins bind to metal surfaces or synthetic polymers. The chemical state of the metal or polymer surface can be directly related to protein adhesion, which is a key factor in the design of implants or drug delivery systems.

Study of Metal-Protein Complexes

XPS is also useful in the study of metal-protein complexes, especially for proteins involved in metal binding, such as metalloproteins or enzymes with metal cofactors. These complexes are critical in many biological processes, from electron transfer in photosynthesis to metal ion homeostasis. By studying the chemical state of the metal center in a protein using XPS, it is possible to learn about its coordination environment and how it interacts with other molecules, which is key to understanding the function of the protein.

Surface Modification of Biomaterials

In the field of biomaterial development, surface modification is often used to improve the biocompatibility, stability or functionality of materials. XPS is often used to analyze the surface chemistry of modified biomaterials. For example, coatings or functional groups introduced to surfaces can be characterized with XPS to confirm the presence of specific chemical groups to ensure that modifications have been successful. This is particularly important in the design of devices such as prosthetics or medical implants, where surface chemistry can influence cell adhesion, immune response, and long-term stability.

Drug Design and Delivery

XPS also has a place in drug design and delivery systems. Drugs often interact with biological membranes or are delivered via nanocarriers, and understanding these interactions at the molecular level is critical to improving efficacy and reducing side effects. XPS can be used to study the surface properties of drug delivery systems, such as liposomes, micelles or nanoparticles, and assess how drugs bind to these surfaces. In addition, surface modifications of drug delivery systems that improve stability or targeted delivery can be analyzed with XPS to ensure that the intended chemical environment is achieved.

Advantages and Limitations of XPS

Comparison of X-ray Photoelectron Spectroscopy (XPS), X-ray Powder Diffraction (XRD), and X-ray Fluorescence (XRF)

In summary, XPS is an invaluable tool in structural biology, providing a wealth of information about the chemical environment of biomolecules at surfaces and interfaces. Its ability to provide detailed insights into protein-surface interactions, metal-protein complexes, and surface modifications of biomaterials makes it a versatile tool for diverse applications in biological research and biomaterial development. Despite its limitations, XPS remains an essential technique for understanding the molecular architecture and behavior of biological systems, especially when combined with other complementary methods.

Creative Biostructure provides three types of protein X-ray crystallography services including gene-to-structure services, custom X-ray crystallography services, and fast-track crystallization services. Samples that can be analyzed include small peptide, membrane protein, antibody-antigen complex, and viral envelope glycoprotein. Our skilled crystallographers work closely with clients to ensure a quick turnaround and deliver accurate, dependable results. Contact us today to learn more about how we can support your research project.

References

1. Dayal G, Jinesh KB. Correlation between oxygen vacancies and neuromorphic properties of pulsed laser-deposited bismuth iron oxide artificial synapses. Appl Phys A. 2023;129(11):777.
2. Stevie FA, Donley CL. Introduction to x-ray photoelectron spectroscopy. Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films. 2020;38(6):063204.