Glycosylation, a prevalent and crucial post-translational modification, plays a pivotal role in regulating protein structure, signal transduction, immune responses, embryonic development, and more. It involves the transfer of monosaccharides or oligosaccharides to proteins by glycosyltransferases, forming glycosidic bonds with amino acid residues on the protein. Glycosylation can occur via N-glycosylation, connecting to the side-chain amino group of asparagine, or O-glycosylation, linking to the hydroxyl group of serine or threonine.
Analyzing the complex structures of glycoproteins has long been a challenge in structural biology. Determining glycosylation sites, deciphering glycan structures, and quantifying relative glycoform abundances fall within the scope of protein glycosylation research. However, due to the myriad of oligosaccharide isomers, diverse monosaccharide compositions and linkage patterns, as well as variances in glycosylation sites, glycosylation modifications exhibit both macroheterogeneity and microheterogeneity. Traditional structural analysis methods like cryo-electron microscopy and X-ray crystallography often struggle to comprehensively characterize glycoproteins.
Mass spectrometry (MS) has emerged as a powerful tool for characterizing protein glycosylation due to its high sensitivity, rapid analysis speed, and minimal sample consumption. MS-based glycomics techniques can accurately confirm glycan structures, protein sequences, post-translational modification types, sites, and relative abundances. However, these methods may not directly provide information on protein higher-order structures and dynamics.
Non-denaturing native mass spectrometry (nMS) offers a promising alternative. It allows proteins to be analyzed in their native state under non-denaturing conditions, preserving their intact structures. This enables the identification of glycan compositions and assists in exploring the structure-function relationships between glycosylation and proteins. Despite its potential, nMS requires high mass spectrometry resolution and struggles with direct identification of monosaccharide structures and their modification sites.
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Non-Denaturing Dynamic Conformationally Resolved Mass Spectrometry
Analyzing the heterogeneity of glycosylation modifications is crucial for a comprehensive understanding of glycoprotein structure and function. Glycomics and glycoproteomics based on mass spectrometry offer a direct and accurate means to decipher both the microheterogeneity and macroheterogeneity of glycans. However, the sample preparation process for these methods is labor-intensive, relies heavily on databases, and lacks the ability to provide information on protein higher-order structures. In contrast, native mass spectrometry (nMS) utilizes gentle, easily ionizable buffer systems, such as ammonium acetate solution, to maintain proteins in their non-denatured state. This approach preserves some non-covalent interactions and enables more comprehensive detection of intact glycoproteins and their complexes. It allows for the integration of glycoprotein structural heterogeneity with the chemical stoichiometry and dynamics of complex interactions, facilitating simultaneous analysis at both the glycan and protein levels. For instance, the cell surface of most mammalian cells is adorned with a glycocalyx, a layer of polysaccharide-protein complexes. Due to the high heterogeneity and flexibility of glycan chains, conventional structural biology approaches often lack effective analytical methods. Recent reports have demonstrated the successful use of nMS technology to reveal the differential modulation of membrane transport proteins and receptor protein profiles by highly heterogeneous glycosylation modifications. Presently, nMS instrumentation can be broadly categorized into two types: those based on Orbitrap mass analyzers, such as the Thermo Scientific Q-ExactiveUHMR, which offers a mass-to-charge ratio range of up to m/z 80,000 and a quadrupole selection range of up to m/z 25,000; and those based on time-of-flight (TOF) analyzers, commonly used in conjunction with ion mobility for two-dimensional gas-phase separation, ultimately enhancing the structural resolution of ion detection and identification.
Ion mobility-mass spectrometry (IM-MS) is a two-dimensional analytical technique that combines ion mobility spectrometry with mass spectrometry. Its fundamental operating principle involves the flight of analyte ions through a drift tube under the influence of an electric field, where they collide with a buffer gas filled in the tube. Due to differences in ion shape, size, and charge, their migration rates vary, resulting in distinct arrival time distributions (ATD) in the ion mobility cell, thereby enabling rapid separation. ATD can be converted into ion structure-related parameters, such as collisional cross section (CCS), using the Mason-Schamp equation, facilitating direct comparative analysis of results obtained from different instruments in various laboratories. IM-MS allows for the monitoring of the dynamic conformational transitions and stability of protein ions through collision-induced unfolding (CIU). Specifically, CIU involves incrementally increasing collision voltage to sequentially activate protein ions, inducing collision voltage-dependent dynamic conformational changes. Subsequent to these conformational alterations, a series of conformers are separated by ion mobility and their ATD or CCS are collected. Visualization software like CIUSuite can then generate fingerprint spectra of the target protein's unfolding. Emerging structural mass spectrometry techniques based on CIU-IM-MS have been increasingly applied in the analysis of glycoprotein conformations. For instance, Robinson's research group utilized this technique to uncover a positive correlation between the number of O-glycosylation modification sites and the stability of the DC-SIGN glycoprotein conformation. Furthermore, advancements in visualization software, such as the second-generation CIUSuite2 developed by Ruotolo's team, have accelerated progress in this direction. This software is compatible with raw data from various commercial instruments and enables the extraction and analysis of multi-charge state ion information, providing rapid insights into protein conformation stability, domain abundance, and binding kinetics.
The protein produced by electrospray ionization is typically present in multiple charge states due to the influence of solution composition and pH. These different charge states may correspond to distinct conformational states of the protein. However, traditional CIU techniques typically analyze only one charge state of the protein at a time. On one hand, this single-charge-state-based analysis mode sacrifices various degrees of throughput, analysis speed, and overall conformational data collection for the protein. On the other hand, for highly heterogeneous and difficult-to-ionize glycoproteins, as the collision voltage increases, there may be a shift in charge states, leading to significant interference and thus larger errors in the analysis results based on a single charge state. Consequently, Phetsanthad et al. developed a novel approach termed All-Ion Unfolding (AIU) to address these challenges. Unlike CIU, AIU omits the quadrupole ion selection process before ion activation and unfolding. Instead, AIU simultaneously activates all protein ions generated by electrospray ionization. To maximize the extraction of all protein conformational information, the researchers also devised a toolkit to simultaneously extract all ion unfolding information and introduced a novel conformational parameter termed CCSacc, which is used to generate conformational unfolding fingerprint spectra independent of charge state. This approach effectively enhances analysis speed and throughput while retaining all protein conformational information. Researchers found that the AIU operating mode could save at least 75% of data acquisition time for most proteins and significantly improve the structural resolution performance for highly heterogeneous glycoproteins.
IM-MS analysis of carbohydrate fragments (Hofmann et al., 2017)
Applications of Non-Denaturing Dynamic Conformational Mass Spectrometry Technology in the Biomedical Field
Drug Discovery and Development:
nMS plays a crucial role in drug discovery and development by providing insights into the structural dynamics of biomolecules involved in drug-target interactions. It enables researchers to elucidate the conformational changes of proteins and their complexes in response to drug binding, offering valuable information for rational drug design and optimization. By studying protein-ligand interactions and conformational changes, nMS facilitates the identification of potential drug candidates and the evaluation of their efficacy and specificity.
Biomarker Discovery:
In biomarker discovery, nMS serves as a powerful tool for identifying and characterizing biomolecules associated with various diseases, including cancer, neurodegenerative disorders, and metabolic diseases. By analyzing the conformational profiles of proteins and peptides in biological samples, nMS enables the identification of disease-specific biomarkers that can be used for early detection, prognosis, and monitoring of disease progression. Additionally, nMS facilitates the study of PTMs and their role in disease pathogenesis, further enhancing our understanding of disease mechanisms.
Disease Diagnosis:
nMS contributes to disease diagnosis by enabling the detection and quantification of disease-related biomolecules in clinical samples, such as blood, urine, and tissue specimens. By analyzing the conformational changes and PTMs of proteins, nMS can differentiate between healthy and diseased states, providing valuable diagnostic information for various conditions. Moreover, nMS-based approaches offer the potential for developing minimally invasive diagnostic tests with high sensitivity and specificity, improving patient outcomes through early and accurate disease detection.
Disease Treatment:
In disease treatment, nMS plays a role in personalized medicine by guiding treatment strategies based on the molecular characteristics of individual patients. By profiling the conformational changes of proteins and their response to therapeutic interventions, nMS facilitates the selection of optimal treatment regimens and monitoring of treatment efficacy. Additionally, nMS contributes to the development of targeted therapies and precision medicine approaches by elucidating the mechanisms of action of therapeutic agents and identifying potential drug resistance mechanisms.
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Assessing Advantages and Limitations
Non-denaturing dynamic conformational mass spectrometry (nDC-MS) technology holds significant advantages in biomedical applications, making it a powerful tool for various analyses. Its strengths lie in its high sensitivity, high resolution, and rapid analysis speed.
One of the primary advantages of nDC-MS is its high sensitivity, which allows for the detection of molecular interactions and conformational changes at low concentrations. This sensitivity is particularly valuable in biomedical research, where subtle changes in protein structure or modifications can have profound implications for disease diagnosis and treatment.
nDC-MS offers high resolution, enabling the precise characterization of complex biomolecular structures. This capability is crucial for identifying specific protein isoforms, elucidating protein-protein interactions, and mapping post-translational modifications, all of which are essential for understanding disease mechanisms and developing targeted therapies.
Another advantage is the rapid analysis speed of nDC-MS, which allows for high-throughput screening of samples. This speed facilitates large-scale studies, such as drug discovery efforts or biomarker identification projects, where the analysis of numerous samples is required to identify potential therapeutic targets or diagnostic markers.
However, despite its many strengths, nDC-MS may have limitations under certain circumstances. For example, the technique often requires meticulous sample preparation procedures to maintain the native structure of biomolecules. Any deviations from optimal conditions can affect the reliability and reproducibility of the results, making sample preparation a critical factor in nDC-MS analysis.
The interpretation of mass spectrometry data generated by nDC-MS can be complex and challenging. Analyzing the vast amount of data produced and accurately interpreting the results require sophisticated computational tools and expertise in mass spectrometry data analysis. This complexity can pose a barrier for researchers without specialized training or access to advanced bioinformatics resources.
In summary, while non-denaturing dynamic conformational mass spectrometry technology offers numerous advantages for biomedical research, including high sensitivity, resolution, and analysis speed, it also presents challenges related to sample preparation requirements and data interpretation complexities. Addressing these limitations through method optimization and advancements in data analysis techniques will be essential for maximizing the utility of nDC-MS in biomedical applications.
Reference
- Hofmann, Johanna, and Kevin Pagel. "Glycan analysis by ion mobility–mass spectrometry." Angewandte Chemie International Edition 56.29 (2017): 8342-8349.
