Folding Process Research Articles

Ab initio characterization of protein molecular dynamics with AI2BMD.

Biomolecular dynamics simulation is a fundamental technology for life sciences research, and its usefulness depends on its accuracy and efficiency1-3. Classical molecular dynamics simulation is fast but lacks chemical accuracy4,5. Quantum chemistry methods such as density functional theory can reach chemical accuracy but cannot scale to support large biomolecules6. Here we introduce an artificial intelligence-based ab initio biomolecular dynamics system (AI2BMD) that can efficiently simulate full-atom large biomolecules with ab initio accuracy. AI2BMD uses a protein fragmentation scheme and a machine learning force field7 to achieve generalizable ab initio accuracy for energy and force calculations for various proteins comprising more than 10,000 atoms. Compared to density functional theory, it reduces the computational time by several orders of magnitude. With several hundred nanoseconds of dynamics simulations, AI2BMD demonstrated its ability to efficiently explore the conformational space of peptides and proteins, deriving accurate 3J couplings that match nuclear magnetic resonance experiments, and showing protein folding and unfolding processes. Furthermore, AI2BMD enables precise free-energy calculations for protein folding, and the estimated thermodynamic properties are well aligned with experiments. AI2BMD could potentially complement wet-lab experiments, detect the dynamic processes of bioactivities and enable biomedical research that is impossible to conduct at present.

Antiparallel G-Quadruplex Formation Hinders Conversion to a Parallel Topology.

G-quadruplexes (G4s) are four-stranded structures formed by guanine-rich sequences. While their structures, properties, and applications have been extensively studied, an understanding of their folding processes remains limited. In this study, we investigated the folding of the sequence d[(G3T2)3G3] in potassium solutions, focusing on the impact of a folding intermediate on the overall folding process. Our results indicate that this sequence eventually folds into a parallel G4 structure, either directly or through an antiparallel conformation intermediate, suggesting the existence of a specific competitive folding process. Detailed kinetic analysis using stopped-flow techniques reveals that the antiparallel conformation forms much faster than the parallel one. This antiparallel G4 slowly converts to the thermodynamically favored parallel topology, thus slowing the overall folding rate. As a result, the formation of the parallel quadruplex via an antiparallel G4 intermediate is slower than the direct process, indicating that this antiparallel conformation negatively impacts the overall folding process in a temperature-dependent manner. Interestingly, sodium was shown to facilitate the conversion from antiparallel to parallel. These analyses highlight the complexity of the G4 folding process, which is crucial for most biological applications.

Preferential binding of ADP-bound mitochondrial HSP70 to the nucleotide exchange factor GRPEL1 over GRPEL2.

Human nucleotide exchange factors GRPEL1 and GRPEL2 play pivotal roles in the ADP-ATP exchange within the protein folding cycle of mitochondrial HSP70 (mtHSP70), a crucial chaperone facilitating protein import into the mitochondrial matrix. Studies in human cells and mice have indicated that while GRPEL1 serves as an essential co-chaperone for mtHSP70, GRPEL2 has a role regulated by stress. However, the precise structural and biochemical mechanisms underlying the distinct functions of the GRPEL proteins have remained elusive. In our study, we present evidence revealing that ADP-bound mtHSP70 exhibits remarkably higher affinity for GRPEL1 compared to GRPEL2, with the latter experiencing a notable decrease in affinity upon ADP binding. Additionally, Pi assay showed that GRPEL1, but not GRPEL2, enhanced the ATPase activity of mtHSP70. Utilizing Alphafold modeling, we propose that the interaction between GRPEL1 and mtHSP70 can induce the opening of the nucleotide binding cleft of the chaperone, thereby facilitating the release of ADP, whereas GRPEL2 lacks this capability. Additionally, our findings suggest that the redox-regulated Cys87 residue in GRPEL2 does not play a role in dimerization but rather reduces its affinity for mtHSP70. Our findings on the structural and functional disparities between GRPEL1 and GRPEL2 may have implications for mitochondrial protein folding and import processes under varying cellular conditions.

Exploring the role of different cell types on cortical folding in the developing human brain through computational modeling

The human brain’s distinctive folding pattern has attracted the attention of researchers from different fields. Neuroscientists have provided insights into the role of four fundamental cell types crucial during embryonic development: radial glial cells, intermediate progenitor cells, outer radial glial cells, and neurons. Understanding the mechanisms by which these cell types influence the number of cortical neurons and the emerging cortical folding pattern necessitates accounting for the mechanical forces that drive the cortical folding process. Our research aims to explore the correlation between biological processes and mechanical forces through computational modeling. We introduce cell-density fields, characterized by a system of advection-diffusion equations, designed to replicate the characteristic behaviors of various cell types in the developing brain. Concurrently, we adopt the theory of finite growth to describe cortex expansion driven by increasing cell density. Our model serves as an adjustable tool for understanding how the behavior of individual cell types reflects normal and abnormal folding patterns. Through comparison with magnetic resonance images of the fetal brain, we explore the correlation between morphological changes and underlying cellular mechanisms. Moreover, our model sheds light on the spatiotemporal relationships among different cell types in the human brain and enables cellular deconvolution of histological sections.

The origin of mutational epistasis.

The interconnected processes of protein folding, mutations, epistasis, and evolution have all been the subject of extensive analysis throughout the years due to their significance for structural and evolutionary biology. The origin (molecular basis) of epistasis-the non-additive interactions between mutations-is still, nonetheless, unknown. The existence of a new perspective on protein folding, a problem that needs to be conceived as an 'analytic whole', will enable us to shed light on the origin of mutational epistasis at the simplest level-within proteins-while also uncovering the reasons why the genetic background in which they occur, a key component of molecular evolution, could foster changes in epistasis effects. Additionally, because mutations are the source of epistasis, more research is needed to determine the impact of post-translational modifications, which can potentially increase the proteome's diversity by several orders of magnitude, on mutational epistasis and protein evolvability. Finally, a protein evolution thermodynamic-based analysis that does not consider specific mutational steps or epistasis effects will be briefly discussed. Our study explores the complex processes behind the evolution of proteins upon mutations, clearing up some previously unresolved issues, and providing direction for further research.

Protein folding: Funnel model revised

The spatial structure of proteins, largely determined by their amino acid sequences, is also dependent on the environmental conditions under which the folding process takes place. In aqueous environments, exposure of polar amino acids is the driving factor, whereas protein stabilization in amphipathic membranes requires exposure to hydrophobic residues. This observation can be extended to all other environmental conditions under which proteins exhibit biological activity and, most importantly, to the folding process. The fuzzy oil drop (FOD) model assumes a centric location of hydrophobic residues (hydrophobic core) with exposure of polar residues towards the aqueous environment, as the influence of the aqueous environment is extended to include the contribution of other non-aqueous factors, enabling the assessment of their influence on protein structuring. The application of the modified FOD model (FOD-M) we have developed allows the environment to be represented as an external force field in the form of a continuum. The role of environmental conditions allows modification of the funnel model expressing the localization of the energy minimum as dependent on external conditions expressed by the K scale, where K measures the degree of other than polar water factors participating in folding process.

High-Fidelity Supramolecular Chirality Transportation Enabled Through Chalcogen Bonding.

The formation of asymmetric microenvironments in proteins benefits from precise transportation of chirality across multiple levels through weak bonds in the folding and assembly process, which inspires the rational design and fabrication of artificial chiral materials. Herein, the chalcogen bonding-directed precise transportation of supramolecular chirality toward multiple levels is reported to aid the fabrication of chiroptical materials. Benzochalcogenadiazole (O, S, Se) motifs are conjugated to amino acid residues, and the solid-state assemblies afforded selective supramolecular chirality with handedness depending on the kinds of chalcogen atoms and amino acids. The chalcogen-N sequence assisted by hydrogen bonding synergistically allows for the complexation with pyrene conjugated aryl carboxylic acids to give macroscopic helical structures with active circular dichroism and circularly polarized luminescence, of which handedness is precisely determined by the pristine chiral species. Then the further application of chiral benzochalcogenadiazole motifs as seeds in directing handedness of benzamide via symmetry breaking is realized. Behaving as the dopants, embedding into the matrix of benzophenone induces the room temperature phosphorescence, whereby the thermal chiroptical switch is fabricated with color-tunable phosphorescent circularly polarized luminescence. This work utilized benzochalcogenadiazole-based chiral building units to accomplish precise transportation of supramolecular chirality in coassemblies with high-fidelity, achieving flexible manipulation of chiroptical properties and macroscopic helical sense.

Pulling Forces Differentially Affect Refolding Pathways Due to Entangled Misfolded States in SARS-CoV-1 and SARS-CoV-2 Receptor Binding Domain

Single-molecule force spectroscopy (SMFS) experiments can monitor protein refolding by applying a small force of a few piconewtons (pN) and slowing down the folding process. Bell theory predicts that in the narrow force regime where refolding can occur, the folding time should increase exponentially with increased external force. In this work, using coarse-grained molecular dynamics simulations, we compared the refolding pathways of SARS-CoV-1 RBD and SARS-CoV-2 RBD (RBD refers to the receptor binding domain) starting from unfolded conformations with and without a force applied to the protein termini. For SARS-CoV-2 RBD, the number of trajectories that fold is significantly reduced with the application of a 5 pN force, indicating that, qualitatively consistent with Bell theory, refolding is slowed down when a pulling force is applied to the termini. In contrast, the refolding times of SARS-CoV-1 RBD do not change meaningfully when a force of 5 pN is applied. How this lack of a Bell response could arise at the molecular level is unknown. Analysis of the entanglement changes of the folded conformations revealed that in the case of SARS-CoV-1 RBD, an external force minimizes misfolding into kinetically trapped states, thereby promoting efficient folding and offsetting any potential slowdown due to the external force. These misfolded states contain non-native entanglements that do not exist in the native state of either SARS-CoV-1-RBD or SARS-CoV-2-RBD. These results indicate that non-Bell behavior can arise from this class of misfolding and, hence, may be a means of experimentally detecting these elusive, theoretically predicted states.

Gust Response and Alleviation of Avian-Inspired In-Plane Folding Wings.

The in-plane folding wing is one of the important research directions in the field of morphing or bionic aircraft, showing the unique application value of enhancing aircraft maneuverability and gust resistance. This article provides a structural realization of an in-plane folding wing and an aeroelasticity modeling method for the folding process of the wing. By approximating the change in structural properties in each time step, a method for calculating the structural transient response expressed in recursive form is obtained. On this basis, an aeroelasticity model of the wing is developed by coupling with the aerodynamic model using the unsteady panel/viscous vortex particle hybrid method. A wind-tunnel test is implemented to demonstrate the controllable morphing capability of the wing under aerodynamic loads and to validate the reliability of the wing loads predicted by the method in this paper. The results of the gust simulation show that the gust scale has a significant effect on the response of both the open- and closed-loop systems. When the gust alleviation controller is enabled, the peak bending moment at the wing root can be reduced by 5.5%∼47.3% according to different gust scales.

Same Day Access to Folded Synthetic Proteins.

Understanding protein function is a cornerstone of modern biology. Research centers worldwide dedicate significant efforts to prepare individual proteins for study, the isolation and purification of which can take days to months. We developed a workflow that enables same-day access to functional synthetic proteins. Chemical synthesis provides access to crude protein chains in hours, but the removal of the synthetic side products is typically a days-long process. We find that chemical modifications on side products lead to significant and unpredictable changes in the folding behavior. Consistent with these findings, we discovered that approaches based on biophysical properties characteristic of the folded protein target can discriminate against chemically similar species. Confirming our protocol with nine protein targets, we show that appropriate desalting followed by different folding strategies enables isolation of functional single-domain proteins in hours instead of days. Each target was isolated in under 10 h, including some proteins with post-translational modifications, non-natural amino acids, and disulfide bonds. Rapid biological discovery requires on-demand access to proteins, and the folding pipeline described here is uniquely suited to enabling these efforts. The folding process presented here was not assessed on complex proteins, and therefore, it may require further optimization in those cases.

‘Invisible’ Molecular Dynamics Revealed for a Conformationally Chiral π‐Stacked Perylene Bisimide Foldamer

Whilst energetic and kinetic aspects of folding processes are meanwhile well understood for natural biomacromolecules, the folding dynamics in so far studied artificial foldamer counterparts remain largely unexplored. This is due to the low energy barriers between their conformational isomers that make the dynamic processes undetectable with conventional methods such as UV/vis absorption, fluorescence, and NMR spectroscopy, making such processes ‘invisible’. Here we present an asymmetric perylene bisimide dimer (bis‐PBI 1) that possesses conformational chirality in its folded state. Owing to the large interconversion barrier (≥ 116 kJ mol–1), four stereoisomers could be separated and isolated. Since the interconversion between these stereoisomers requires the foldamer to first open and then to re‐fold, the transformation of one stereoisomer into others allowed us to ‘visualize’ the dynamics of folding with time and determine its lifetimes and the energetic barriers associated with the folding process. Supported by quantum chemical calculations, we identified the open structure to be only a fleeting metastable state of higher energy. Our experimental observation of the kinetics associated with the molecular dynamics in the PBI foldamer advances the fundamental understanding of folding in synthetic foldamers and paves the way for the design of smart functional materials.

'Invisible' Molecular Dynamics Revealed for a Conformationally Chiral π-Stacked Perylene Bisimide Foldamer.

Whilst energetic and kinetic aspects of folding processes are meanwhile well understood for natural biomacromolecules, the folding dynamics in so far studied artificial foldamer counterparts remain largely unexplored. This is due to the low energy barriers between their conformational isomers that make the dynamic processes undetectable with conventional methods such as UV/vis absorption, fluorescence, and NMR spectroscopy, making such processes 'invisible'. Here we present an asymmetric perylene bisimide dimer (bis-PBI 1) that possesses conformational chirality in its folded state. Owing to the large interconversion barrier (≥ 116 kJ mol-1), four stereoisomers could be separated and isolated. Since the interconversion between these stereoisomers requires the foldamer to first open and then to re-fold, the transformation of one stereoisomer into others allowed us to 'visualize' the dynamics of folding with time and determine its lifetimes and the energetic barriers associated with the folding process. Supported by quantum chemical calculations, we identified the open structure to be only a fleeting metastable state of higher energy. Our experimental observation of the kinetics associated with the molecular dynamics in the PBI foldamer advances the fundamental understanding of folding in synthetic foldamers and paves the way for the design of smart functional materials.

Enlarged Curvature, Torsion and Torque in Helical Conformations and the Stability and Growth of α-Peptide under the Isochoric and Isobaric Conditions: Variatonal Optimization

The torsional deformation behavior of an elastic bar with a circular cross-section was investigated by applying invariant dyadic analysis, where the small finite displacement functions advocated by Saint-Venant (1855) were fully employed. It was found that the previously overlooked circumferential shear force field generated by pure torsion on the side walls of a bar produces an unusual torque term induced by the skew-symmetric part of the deformation tensor and exhibits quadratic length dependence along the z-axis of the bar. The adaptation of this torque term for a helical conformation of α-peptides creates moments acting on the circular cross-sections and is directed along the surface normal of circular cross-sections, which coincides with the tangent vector of the helix. The projection of this torque along the z-axis of the helix varies quadratically with the azimuthal angle. The radial component of the unusual torque, which also lies along the principal normal vector of the helix, starts to perform a precession motion by tracking a spiral orbit around the z-axis, whereas its apex angle decreases asymptotically with the azimuthal angle and finally reaches a finite value depending on the height of the helix along the z-axis. The ordinary torque terms, which are also deduced from the self- and anti-self-conjugate parts of the deformation tensor, have magnitudes half that of the full torque term reported in the literature. The present results were applied to the helical conformation of α-peptides designated by {3.611} to show that the mechanical stability of strained open-ended helical conformations can be successfully achieved by spontaneous readjustments of the surface and bulk Helmholtz free energies under isothermal isochoric conditions. It has been demonstrated that the main contribution to the mechanical stability of α-peptide 3.611 cannot come alone from the electrostatic dipole-dipole interaction potential of the anti-align excess dipole pairs but also from the surface Helmholtz free energy, which is characterized by a binding free energy of -15.5 eV/molecule (-32.56 Kcal/mole) for an alpha-peptide composed of 11 amino acid residues with a critical arc length of approximately 10 nm, assuming that the shear modulus is G = 1GPa and the surface Helmholtz specific free energy density is fs = 800 erg/cm2. This result was in excellent agreement with the experimental observations of the AH-1 conformation of (Glu)n Cys at pH 8. The present theory indicates that only two excess permanent anti-align dipole pairs for one α-Helical peptide molecule is requirement to stabilize the whole secondary structure of the protein that is exposed to heavy torsional deformation during the folding processes which amounts to 7.75 eV/molecule stored electrostatic energy compared to the interfacial Helmholtz free energy of -23.25 eV/molecule, which is exposed to hydrophobic environments.

KDELR2 is necessary for chronic obstructive pulmonary disease airway Mucin5AC hypersecretion via an IRE1α/XBP-1s-dependent mechanism.

Airway mucus hypersecretion, a crucial pathological feature of chronic obstructive pulmonary disease (COPD), contributes to the initiation, progression, and exacerbation of this disease. As a macromolecular mucin, the secretory behaviour of Mucin5AC (MUC5AC) is highly dependent on a series of modifying and folding processes that occur in the endoplasmic reticulum (ER). In this study, we focused on the ER quality control protein KDEL receptor (KDELR) and demonstrated that KDELR2 and MUC5AC were colocalized in the airway epithelium of COPD patients and COPD model rats.In addition, knockdown of KDELR2 markedly reduced the expression of MUC5AC both invivo and invitro and knockdown of ATF6 further decreased the levels of KDELR2. Furthermore, pretreatment with 4μ8C, an IRE1α inhibitor, led to a partial reduction in the expression of KDELR2 and MUC5AC both invivo and invitro, which indicated the involvement of IRE1α/XBP-1s in the upstream signalling cascade. Our study revealed that KDELR2 plays a crucial role in airway MUC5AC hypersecretion in COPD, which might be dependent on ATF6 and IRE1α/XBP-1s upstream signalling.

Kinematics and dynamics characteristics of a double-ring truss deployable antenna mechanism based on triangular prism deployable unit

In order to effectively improve the single-ring truss deployable antenna mechanism due to the large aperture caused by the problem with low structural strength and low-profile accuracy, a series of double-ring truss deployable antenna mechanisms (DRTDAM) are proposed with constant height during folding and deployment process. First, a variety of DRTDAMs are proposed based on tetrahedral units and their topologies are analyzed. Secondly, degree-of-freedom (DOF) characteristics of DRTDAM proposed in this paper are analyzed based on the screw theory and screw-constrained topological graphs and based on this, the kinematic characteristics of DRTDAM is investigated. Thirdly, the dynamics of the whole DRTDAM is built with Newton-Euler equations of multi-rigid body system. Finally, the correctness of above analysis is verified through dynamics analysis software ADAMS and numerical analysis software MATLAB, and the principle prototype is produced to verify the correctness of DOF analysis. The mechanism proposed in this paper enriches the configuration of DRTDAM, and the process of kinematic characterization method is clear and simple, which is meaningful for the research in space complex mechanism.

Implementation of Raita Algorithm in Manado-Indonesia Translation Application with Text Suggestion Using Levenshtein Distance Algorithm

Abstract. Manado City is one of the multidimensional and multicultural cities, possessing assets that are considered highly potential for development into tourism and development attractions. The current tourism assets being developed by the Manado City government are cultural tourism, as they hold a charm and allure for tourists. Hence, a communication tool in the form of a translation application is necessary for facilitating communication between visiting tourists and the native community of North Sulawesi, even for newcomers who intend to reside in North Sulawesi, given that the Manado language serves as the primary communication tool within the community. This research employs a combination of the Raita algorithm and the Levenshtein distance algorithm in its creation process, along with the confusion matrix method to calculate the accuracy of translation results using the Levenshtein distance algorithm with a text suggestion feature. The research begins by collecting a dataset consisting of Manado language vocabulary and their translations in Indonesia language, sourced from literature studies and original respondents from North Sulawesi, which have been validated by a validator to prevent translation data errors. The subsequent stage involves preprocessing the dataset, converting the entire content of the dataset to lowercase using the case folding process, and removing spaces at the start and end of texts using the trim function. Next, both algorithms are implemented, with the Raita algorithm serving for translation and the Levenshtein distance algorithm providing text suggestions for typing errors during the translation process. The accuracy results derived from the confusion matrix calculations during the translation process of 100 vocabulary words, accounting for typing errors, indicate that the Levenshtein distance algorithm is capable of effectively translating vocabulary accurately and correctly, even in the presence of typing errors, resulting in a high accuracy rate of 94,17%. Purpose: To determine the implementation of the Levenshtein distance and Raita algorithms in the process of using the Manado-Indonesian translation application, as well as the resulting accuracy level. Methods/Study design/approach: In this study, a combination of the Raita and Levenshtein distance algorithms is utilized in the translation application system, along with the confusion matrix method to calculate accuracy. Result/Findings: The accuracy achieved in the translation process using text suggestions from the Levenshtein distance algorithm is 94.17%. Novelty/Originality/Value: This research demonstrates that the combination of the Raita and Levenshtein distance algorithms yields optimal results in the vocabulary translation process and provides accurate outcomes from the use of effective text suggestions. This is attributed to the fact that nearly all the data used was successfully translated by the system, even in the presence of typographical errors.

Spectral Map for Slow Collective Variables, Markovian Dynamics, and Transition State Ensembles.

Understanding the behavior of complex molecular systems is a fundamental problem in physical chemistry. To describe the long-time dynamics of such systems, which is responsible for their most informative characteristics, we can identify a few slow collective variables (CVs) while treating the remaining fast variables as thermal noise. This enables us to simplify the dynamics and treat it as diffusion in a free-energy landscape spanned by slow CVs, effectively rendering the dynamics Markovian. Our recent statistical learning technique, spectral map [Rydzewski, J. J. Phys. Chem. Lett. 2023, 14(22), 5216-5220], explores this strategy to learn slow CVs by maximizing a spectral gap of a transition matrix. In this work, we introduce several advancements into our framework, using a high-dimensional reversible folding process of a protein as an example. We implement an algorithm for coarse-graining Markov transition matrices to partition the reduced space of slow CVs kinetically and use it to define a transition state ensemble. We show that slow CVs learned by spectral map closely approach the Markovian limit for an overdamped diffusion. We demonstrate that coordinate-dependent diffusion coefficients only slightly affect the constructed free-energy landscapes. Finally, we present how spectral maps can be used to quantify the importance of features and compare slow CVs with structural descriptors commonly used in protein folding. Overall, we demonstrate that a single slow CV learned by spectral map can be used as a physical reaction coordinate to capture essential characteristics of protein folding.

Chameleon Sequences-Structural Effects in Proteins Characterized by Hydrophobicity Disorder.

Repeated protein folding processes both in vivo and in vitro leading to the same structure for a specific amino acid sequence prove that the amino acid sequence determines protein structuring. This is also evidenced by the variability of structuring, dependent on the introduced mutations. An important phenomenon in this regard is the presence of a differentiated secondary structure for chain fragments of identical sequence representing distinct forms of the secondary-order structure. Proteins termed chameleon proteins contain polypeptide chain fragments of identical sequence (length 6-12 aa) showing structural differentiation: helix versus β-structure. In the present paper, it was shown that these fragments represent components matching the structural status dictated by the physicochemical properties of the entire structural unit. This structural matching is related to achieving the goal of the biological function of the structural unit. The corresponding secondary structure represents a means to achieving this goal, not an end in itself. A selected set of proteins from the ChSeq database have been analyzed using a fuzzy oil drop model (FOD-M) identifying the uniqueness of the hydrophobicity distribution taken as a medium for recording the specificity of a given protein and a given chameleon section in particular. It was shown that in the vast majority, the status of chameleon sections turns out to be comparable regardless of the represented secondary structure.

Unsupervised learning of progress coordinates during weighted ensemble simulations: Application to millisecond protein folding.

Our method identifies outliers in a latent space model of the system's sampled conformations that is periodically trained using a convolutional variational autoencoder. As a proof of principle, we applied our DL-enhanced WE method to simulate a millisecond protein folding process. To enable rapid tests, our simulations propagated discrete-state synthetic molecular dynamics trajectories using a generative, fine-grained Markov state model. Results revealed that our ″on-the-fly″ DL of outliers enhanced the efficiency of WE by >3-fold in estimating the folding rate constant. Our efforts are a significant step forward in the unsupervised learning of slow coordinates during rare event sampling.

Acute-phase plasma proteomics of rabbit lung VX2 tumors treated by image-guided microwave ablation.

To explore the plasma proteomic changes of rabbit lung VX2 tumors treated by microwave ablation, and to explore the molecular pathway mechanisms that may be involved. New Zealand white rabbits were inoculated with VX2 tumor cell suspension in the right lower lung and treated with microwave ablation after 2-3 weeks of tumor formation. Blood was collected at 5 time points (TP1~TP5) before and after ablation by cardiac blood sampling and pre-treated before proteomic analysis. The plasma proteome was analyzed by Data-Independent Acquisition (DIA). Different molecular pathways were activated at different time points:(i) TP1vsTP2: more proteins were down-regulated and enrichment analysis showed that the proteasome pathway was activated. The abnormal protein folding process involved in this pathway is closely related to the process of tumor development. (ii) TP2vsTP3: more proteins were up-regulated although the number of differentially differentiated proteins was lower and enrichment analysis showed that the phagosome pathway was activated. After microwave ablation inactivates tumor cells, it activates the phagosomal pathway for immune clearance of necrotic tumor tissue. (iii) TP3vsTP4: more down-regulated proteins, enrichment analysis showed that cysteine and methionine metabolism pathway was activated. Decreased metabolism of these amino acids suggests that cancer progression may be blocked after microwave ablation therapy. (iv) TP4vsTP5: the number of differential proteins was less and more down-regulated proteins, enrichment analysis showed that glutathione metabolism and metabolism of xenobiotics by cytochrome P450 pathway were activated. The down-regulated proteins in this pathway may suggest that microwave ablation may have reduced resistance to certain chemotherapeutic agents following. In the process of lung cancer treatment by microwave ablation, the changes of proteins on the possible molecular pathways at each time point are related to lung cancer, and not only involve some simple inflammatory reactions, and some of the proteins released by destroying the tumor cells can be used as possible drug binding sites and reduce drug resistance.