The role of interleukin-10 in mitigating endoplasmic reticulum stress in aged mice through exercise.

Although unfolded protein response (UPR) is essential for cellular protection, its prolonged activation may induce apoptosis, compromising cellular longevity. The aging process increases the endoplasmic reticulum (ER) stress in skeletal muscle. However, whether combined exercise can prevent age-induced ER stress in skeletal muscle remains unknown. Evidence suggests that ER stress may increase inflammation by counteracting the positive effects of interleukin-10 (IL-10), whereas its administration in cells inhibits ER stress and apoptosis. This study verified the effects of aging and combined exercise on physical performance, ER stress markers, and inflammation in the quadriceps of mice. Moreover, we verified the effects of IL-10 on ER stress markers. C57BL/6 mice were distributed into young (Y, 6 mo old), old sedentary (OS, sedentary, 24 mo old), and old trained group (OT, submitted to short-term combined exercise, 24 mo old). To clarify the role of IL-10 in UPR pathways, knockout mice lacking IL-10 were used. The OS mice presented worse physical performance and higher ER stress-related proteins, such as C/EBP homologous protein (CHOP) and phospho-eukaryotic translation initiation factor 2 alpha (p-eIF2α/eIF2α). The exercise protocol increased muscle strength and IL-10 protein levels in OT while inducing the downregulation of CHOP protein levels compared with OS. Furthermore, mice lacking IL-10 increased BiP, CHOP, and p-eIF2α/eIF2α protein levels, indicating this cytokine can regulate the ER stress response in skeletal muscle. Bioinformatics analysis showed that endurance and resistance training downregulated DNA damage inducible transcript 3 (DDIT3) and XBP1 gene expression in the vastus lateralis of older people, reinforcing our findings. Thus, combined exercise is a potential therapeutic intervention for promoting adjustments in ER stress markers in aged skeletal muscle.NEW & NOTEWORTHY Aging elevates endoplasmic reticulum (ER) stress in skeletal muscle, potentially heightening inflammation by opposing interleukin-10 (IL-10) effects. This study found that short-term combined exercise boosted strength and IL-10 protein levels while reducing CHOP protein levels in older mice. In addition, IL-10-deficient mice exhibited increased ER stress markers, highlighting IL-10's role in regulating ER stress in skeletal muscle. Consequently, combined exercise emerges as a therapeutic intervention to elevate IL-10 and adjust ER stress markers in aging.

Reassessing the exon-foldon correspondence using frustration analysis.

Protein folding and evolution are intimately linked phenomena. Here, we revisit the concept of exons as potential protein folding modules across a set of 38 abundant and conserved protein families. Taking advantage of genomic exon-intron organization and extensive protein sequence data, we explore exon boundary conservation and assess the foldon-like behavior of exons using energy landscape theoretic measurements. We found deviations in the exon size distribution from exponential decay indicating selection in evolution. We show that when taken together there is a pronounced tendency to independent foldability for segments corresponding to the more conserved exons, supporting the idea of exon-foldon correspondence. While 45% of the families follow this general trend when analyzed individually, there are some families for which other stronger functional determinants, such as preserving frustrated active sites, may be acting. We further develop a systematic partitioning of protein domains using exon boundary hotspots, showing that minimal common exons correspond with uninterrupted alpha and/or beta elements for the majority of the families but not for all of them.

Alterations in Proteostasis Mechanisms in Niemann-Pick Type C Disease.

Niemann-Pick Type C (NPC) represents an autosomal recessive disorder with an incidence rate of 1 in 150,000 live births, classified within lysosomal storage diseases (LSDs). The abnormal accumulation of unesterified cholesterol characterizes the pathophysiology of NPC. This phenomenon is not unique to NPC, as analogous accumulations have also been observed in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. Interestingly, disturbances in the folding of the mutant protein NPC1 I1061T are accompanied by the aggregation of proteins such as hyperphosphorylated tau, α-synuclein, TDP-43, and ß-amyloid peptide. These accumulations suggest potential disruptions in proteostasis, a regulatory process encompassing four principal mechanisms: synthesis, folding, maintenance of folding, and protein degradation. The dysregulation of these processes leads to excessive accumulation of abnormal proteins that impair cell function and trigger cytotoxicity. This comprehensive review delineates reported alterations across proteostasis mechanisms in NPC, encompassing changes in processes from synthesis to degradation. Additionally, it discusses therapeutic interventions targeting pharmacological facets of proteostasis in NPC. Noteworthy among these interventions is valproic acid, a histone deacetylase inhibitor (HDACi) that modulates acetylation during NPC1 synthesis. In addition, various therapeutic options addressing protein folding modulation, such as abiraterone acetate, DHBP, calnexin, and arimoclomol, are examined. Additionally, treatments impeding NPC1 degradation, exemplified by bortezomib and MG132, are explored as potential strategies. This review consolidates current knowledge on proteostasis dysregulation in NPC and underscores the therapeutic landscape targeting diverse facets of this intricate process.

Exploring the folding landscape of leptin: Insights into threading pathways.

The discovery of new protein topologies with entanglements and loop-crossings have shown the impact of local amino acid arrangement and global three-dimensional structures. This phenomenon plays a crucial role in understanding how protein structure relates to folding and function, affecting the global stability, and biological activity. Protein entanglements encompassing knots and non-trivial topologies add complexity to their folding free energy landscapes. However, the initial native contacts driving the threading event for entangled proteins remains elusive. The Pierced Lasso Topology (PLT) represents an entangled topology where a covalent linker creates a loop in which the polypeptide backbone is threaded through. Compared to true knotted topologies, PLTs are simpler topologies where the covalent-loop persists in all conformations. In this work, the PLT protein leptin, is used to visualize and differentiate the preference for slipknotting over plugging transition pathways along the folding route. We utilize the Energy Landscape Visualization Method (ELViM), a multidimensional projection technique, to visualize and distinguish early threaded conformations that cannot be observed in an in vitro experiment. Critical contacts for the leptin threading mechanisms were identified where the competing pathways are determined by the formation of a hairpin loop in the unfolded basin. Thus, prohibiting the dominant slipknotting pathway. Furthermore, ELViM offers insights into distinct folding pathways associated with slipknotting and plugging providing a novel tool for de novo design and in vitro experiments with residue specific information of threading events in silico.

Sequence-dependent and -independent information in a combined random energy model for protein folding and coding.

Random energy models (REMs) provide a simple description of the energy landscapes that guide protein folding and evolution. The requirement of a large energy gap between the native structure and unfolded conformations, considered necessary for cooperative, protein-like, folding behavior, indicates that proteins differ markedly from random heteropolymers. It has been suggested, therefore, that natural selection might have acted to choose nonrandom amino acid sequences satisfying this particular condition, implying that a large fraction of possible, unselected random sequences, would not fold to any structure. From an informational perspective, however, this scenario could indicate that protein structures, regarded as messages to be transmitted through a communication channel, would not be efficiently encoded in amino acid sequences, regarded as the communication channel for this transmission, since a large fraction of possible channel states would not be used. Here, we use a combined REM for conformations and sequences, with previously estimated parameters for natural proteins, to explore an alternative possibility in which the appropriate shape of the landscape results mainly from the deviation from randomness of possible native structures instead of sequences. We observe that this situation emerges naturally if the distribution of conformational energies happens to arise from two independent contributions corresponding to sequence-dependent and -independent terms. This construction is consistent with the hypothesis of a protein burial folding code, with native structures being determined by a modest amount of sequence-dependent atomic burial information with sequence-independent constraints imposed by unspecific hydrogen bond formation. More generally, an appropriate combination of sequence-dependent and -independent information accommodates the possibility of an efficient structural encoding with the main physical requirement for folding, providing possible insight not only on the folding process but also on several aspects sequence evolution such as neutral networks, conformational coverage, and de novo gene emergence.

Structural characterization of the human DjC20/HscB cochaperone in solution.

J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.

Exploring the structural acrobatics of fold-switching proteins using simplified structure-based models.

Metamorphic proteins are a paradigm of the protein folding process, by encoding two or more native states, highly dissimilar in terms of their secondary, tertiary, and even quaternary structure, on a single amino acid sequence. Moreover, these proteins structurally interconvert between these native states in a reversible manner at biologically relevant timescales as a result of different environmental cues. The large-scale rearrangements experienced by these proteins, and their sometimes high mass interacting partners that trigger their metamorphosis, makes the computational and experimental study of their structural interconversion challenging. Here, we present our efforts in studying the refolding landscapes of two quintessential metamorphic proteins, RfaH and KaiB, using simplified dual-basin structure-based models (SBMs), rigorously footed on the energy landscape theory of protein folding and the principle of minimal frustration. By using coarse-grained models in which the native contacts and bonded interactions extracted from the available experimental structures of the two native states of RfaH and KaiB are merged into a single Hamiltonian, dual-basin SBM models can be generated and savvily calibrated to explore their fold-switch in a reversible manner in molecular dynamics simulations. We also describe how some of the insights offered by these simulations have driven the design of experiments and the validation of the conformational ensembles and refolding routes observed using this simple and computationally efficient models.

Protein nanocondensates: the next frontier.

Over the past decade, myriads of studies have highlighted the central role of protein condensation in subcellular compartmentalization and spatiotemporal organization of biological processes. Conceptually, protein condensation stands at the highest level in protein structure hierarchy, accounting for the assembly of bodies ranging from thousands to billions of molecules and for densities ranging from dense liquids to solid materials. In size, protein condensates range from nanocondensates of hundreds of nanometers (mesoscopic clusters) to phase-separated micron-sized condensates. In this review, we focus on protein nanocondensation, a process that can occur in subsaturated solutions and can nucleate dense liquid phases, crystals, amorphous aggregates, and fibers. We discuss the nanocondensation of proteins in the light of general physical principles and examine the biophysical properties of several outstanding examples of nanocondensation. We conclude that protein nanocondensation cannot be fully explained by the conceptual framework of micron-scale biomolecular condensation. The evolution of nanocondensates through changes in density and order is currently under intense investigation, and this should lead to the development of a general theoretical framework, capable of encompassing the full range of sizes and densities found in protein condensates.

Protein structure prediction from the complementary science perspective.

A comparative analysis between two problems-apparently unrelated-which are solved in a period of ~400 years, viz., the accurate prediction of both the planetary orbits and the protein structures, leads to inferred conjectures that go far beyond the existence of a common path in their resolution, i.e., observation → pattern recognition → modeling. The preliminary results from this analysis indicate that complementary science, together with a new perspective on protein folding, may help us discover common features that could contribute to a more in-depth understanding of still-unsolved problems such as protein folding.

Rethinking the protein folding problem from a new perspective.

One of the main concerns of Anfinsen was to reveal the connection between the amino-acid sequence and their biologically active conformation. This search gave rise to two crucial questions in structural biology, namely, why the proteins fold and how a sequence encodes its folding. As to the why, he proposes a plausible answer, namely, the thermodynamic hypothesis. As to the how, this remains an unsolved challenge. Consequently, the protein folding problem is examined here from a new perspective, namely, as an 'analytic whole'. Conceiving the protein folding in this way enabled us to (i) examine in detail why the force-field-based approaches have failed, among other purposes, in their ability to predict the three-dimensional structure of a protein accurately; (ii) propose how to redefine them to prevent these shortcomings, and (iii) conjecture on the origin of the state-of-the-art numerical-methods success to predict the tridimensional structure of proteins accurately.

Hsp90 and metal-binding J-protein family chaperones are not critically involved in cellular iron-sulfur protein assembly and iron regulation in yeast.

Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions.

High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage.

Environmental stressors, such as temperature, are relevant factors that could generate a negative effect on several tissues in fish. A key fish species for Chilean aquaculture diversification is the red cusk-eel (Genypterus chilensis), a native fish for which knowledge on environmental stressors effects is limited. This study evaluated the effects of high-temperature stress on the liver of red cusk-eel in control (14 °C) and high-temperature (19 °C) groups using multiple approaches: determination of plasmatic hepatic enzymes (ALT, AST, and AP), oxidative damage evaluation (AP sites, lipid peroxidation, and carbonylated proteins), and RNA-seq analysis. High-temperature stress generated a significant increase in hepatic enzyme activity in plasma. In the liver, a transcriptional regulation was observed, with 1239 down-regulated and 1339 up-regulated transcripts. Additionally, high-temperature stress generated oxidative stress in the liver, with oxidative damage and transcriptional modulation of the antioxidant response. Furthermore, an unfolded protein response was observed, with several pathways enriched, as well as a heat shock response, with several heat shock proteins up regulated, suggesting candidate biomarkers (i.e., serpinh1) for thermal stress evaluation in this species. The present study shows that high-temperature stress generated a major effect on the liver of red cusk-eel, knowledge to consider for the aquaculture and fisheries of this species.

Protein conformation and biomolecular condensates.

Protein conformation and cell compartmentalization are fundamental concepts and subjects of vast scientific endeavors. In the last two decades, we have witnessed exciting advances that unveiled the conjunction of these concepts. An avalanche of studies highlighted the central role of biomolecular condensates in membraneless subcellular compartmentalization that permits the spatiotemporal organization and regulation of myriads of simultaneous biochemical reactions and macromolecular interactions. These studies have also shown that biomolecular condensation, driven by multivalent intermolecular interactions, is mediated by order-disorder transitions of protein conformation and by protein domain architecture. Conceptually, protein condensation is a distinct level in protein conformational landscape in which collective folding of large collections of molecules takes place. Biomolecular condensates arise by the physical process of phase separation and comprise a variety of bodies ranging from membraneless organelles to liquid condensates to solid-like conglomerates, spanning lengths from mesoscopic clusters (nanometers) to micrometer-sized objects. In this review, we summarize and discuss recent work on the assembly, composition, conformation, material properties, thermodynamics, regulation, and functions of these bodies. We also review the conceptual framework for future studies on the conformational dynamics of condensed proteins in the regulation of cellular processes.

Evolution and folding of repeat proteins.

Repeat proteins are made with tandem copies of similar amino acid stretches that fold into elongated architectures. These proteins constitute excellent model systems to investigate how evolution relates to structure, folding, and function. Here, we propose a scheme to map evolutionary information at the sequence level to a coarse-grained model for repeat-protein folding and use it to investigate the folding of thousands of repeat proteins. We model the energetics by a combination of an inverse Potts-model scheme with an explicit mechanistic model of duplications and deletions of repeats to calculate the evolutionary parameters of the system at the single-residue level. These parameters are used to inform an Ising-like model that allows for the generation of folding curves, apparent domain emergence, and occupation of intermediate states that are highly compatible with experimental data in specific case studies. We analyzed the folding of thousands of natural Ankyrin repeat proteins and found that a multiplicity of folding mechanisms are possible. Fully cooperative all-or-none transitions are obtained for arrays with enough sequence-similar elements and strong interactions between them, while noncooperative element-by-element intermittent folding arose if the elements are dissimilar and the interactions between them are energetically weak. Additionally, we characterized nucleation-propagation and multidomain folding mechanisms. We show that the global stability and cooperativity of the repeating arrays can be predicted from simple sequence scores.

Coevolution-derived native and non-native contacts determine the emergence of a novel fold in a universally conserved family of transcription factors.

The NusG protein family is structurally and functionally conserved in all domains of life. Its members directly bind RNA polymerases and regulate transcription processivity and termination. RfaH, a divergent sub-family in its evolutionary history, is known for displaying distinct features than those in NusG proteins, which allows them to regulate the expression of virulence factors in enterobacteria in a DNA sequence-dependent manner. A striking feature is its structural interconversion between an active fold, which is the canonical NusG three-dimensional structure, and an autoinhibited fold, which is distinctively novel. How this novel fold is encoded within RfaH sequence to encode a metamorphic protein remains elusive. In this work, we used publicly available genomic RfaH protein sequences to construct a complete multiple sequence alignment, which was further augmented with metagenomic sequences and curated by predicting their secondary structure propensities using JPred. Coevolving pairs of residues were calculated from these sequences using plmDCA and GREMLIN, which allowed us to detect the enrichment of key metamorphic contacts after sequence filtering. Finally, we combined our coevolutionary predictions with molecular dynamics to demonstrate that these interactions are sufficient to predict the structures of both native folds, where coevolutionary-derived non-native contacts may play a key role in achieving the compact RfaH novel fold. All in all, emergent coevolutionary signals found within RfaH sequences encode the autoinhibited and active folds of this protein, shedding light on the key interactions responsible for the action of this metamorphic protein.

Improving coarse-grained models of protein folding through weighting of polar-polar/hydrophobic-hydrophobic interactions into crowded spaces.

Herein were tested 7 hydrophobic-polar sequences in two types of 2D-square space lattices, homogeneous and correlated, the latter simulating molecular crowding included as a geometric boundary restriction. Optimization of 2D structures was carried out using a variant of Dill's model, inspired by convex function, taking into account both hydrophobic (Dill's model) and polar interactions, including more structural information to reach better folding solutions. While using correlated networks, degrees of freedom in the folding of sequences were limited; as a result in all cases, more successful structural trials were found in comparison to a homogeneous lattice. The majority of employed sequences were designed by our workgroup, two of them were folded with other approaches, and another is a modified version of a previous sequence, initial forms of the other two have been employed but without taking into account polar-polar contributions. Three of them are newly proposed, intended to test the conjoint hydrophobic-hydrophobic and polar-polar contributions in crowded spaces. One sequence turned out to be the most difficult of the seven folded, this perhaps due to intrinsic (i) degrees of freedom and (ii) motifs of the expected 2D HP structure. Meanwhile two-sequence, although optimal folding was not achieved for neither of the two approaches, folding with correlated network approach not only produced better results than homogeneous space, but for them the best values found with crowding were very close to the expected optimal fitness. In general, five sequences were better folded with medium lattice units for correlated media; instead, another two sequences were better folded with a bit larger degree of lattice unit, revealing that depending on the degrees of freedom and particular folding, motifs in each sequence would require tuned crowding to achieve better folding. Therefore, the main goal herein was to obtain a modified 2D HP lattice model to mimic folding of proteins or secondary structures, like ß-sheets, taking into account both hydrophobic-hydrophobic and polar-polar interactions, and fold them in a crowded environment. This simple but enough construction would be conducted to determine the needed information to fold sequences in a sort of a minimal but complete heuristic model. Finally, we claim that all folded sequences into crowded spaces achieve better results than homogeneous ones.

Resolving the fine structure in the energy landscapes of repeat proteins.

Ankyrin (ANK) repeat proteins are coded by tandem occurrences of patterns with around 33 amino acids. They often mediate protein-protein interactions in a diversity of biological systems. These proteins have an elongated non-globular shape and often display complex folding mechanisms. This work investigates the energy landscape of representative proteins of this class made up of 3, 4 and 6 ANK repeats using the energy-landscape visualisation method (ELViM). By combining biased and unbiased coarse-grained molecular dynamics AWSEM simulations that sample conformations along the folding trajectories with the ELViM structure-based phase space, one finds a three-dimensional representation of the globally funnelled energy surface. In this representation, it is possible to delineate distinct folding pathways. We show that ELViMs can project, in a natural way, the intricacies of the highly dimensional energy landscapes encoded by the highly symmetric ankyrin repeat proteins into useful low-dimensional representations. These projections can discriminate between multiplicities of specific parallel folding mechanisms that otherwise can be hidden in oversimplified depictions.

Localization of Energetic Frustration in Proteins.

We present a detailed heuristic method to quantify the degree of local energetic frustration manifested by protein molecules. Current applications are realized in computational experiments where a protein structure is visualized highlighting the energetic conflicts or the concordance of the local interactions in that structure. Minimally frustrated linkages highlight the stable folding core of the molecule. Sites of high local frustration, in contrast, often indicate functionally relevant regions such as binding, active, or allosteric sites.

The glycine betaine role in neurodegenerative, cardiovascular, hepatic, and renal diseases: Insights into disease and dysfunction networks.

Glycine betaine (N, N, N-trimethyl amine) is an osmolyte accumulated in cells that is key for cell volume and turgor regulation, is the principal methyl donor in the methionine cycle and is a DNA and proteins stabilizer. In humans, glycine betaine is synthesized from choline and can be obtained from some foods. Glycine betaine (GB) roles are illustrated in chemical, metabolic, agriculture, and clinical medical studies due to its chemical and physiological properties. Several studies have extensively described GB role and accumulation related to specific pathologies, focusing mainly on analyzing its positive and negative role in these pathologies. However, it is necessary to explain the relationship between glycine betaine and different pathologies concerning its role as an antioxidant, ability to methylate DNA, interact with transcription factors and cell receptors, and participate in the control of homocysteine concentration in liver, kidney and brain. This review summarizes the most important findings and integrates GB role in neurodegenerative, cardiovascular, hepatic, and renal diseases. Furthermore, we discuss GB impact on other dysfunctions as inflammation, oxidative stress, and glucose metabolism, to understand their cross-talks and provide reliable data to establish a base for further investigations.

Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay.

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of formerly N-glycosylated peptides (FNGPs). LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases. SIGNIFICANCE: We describe a novel method termed limited deglycosylation assay (LDA), to probe conformational changes of glycoproteins on a systems-wide scale. This method improves the current toolbox of structural proteomics by combining site and conformational-specific PNGase F enzymatic activity with large scale quantitative proteomics. X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoEM techniques are the major techniques applied to elucidate macromolecule structures. However, the size and heterogeneity of the oligosaccharide chains poses several challenges to the applications of these techniques to glycoproteins. The LDA method presented here, can be applied to a range of pathophysiological conditions and expanded to investigate PTMs-mediated structural changes in complex proteomes.