Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles.

Introduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered. Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet-visual spectroscopy, interferometric light microscopy and liquid chromatography-mass spectrometry. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.

On-DNA Synthesis of Multisubstituted Indoles.

The increasing role of the DNA-encoded library technology in early phase drug discovery represents a significant demand for DNA-compatible synthetic methods for therapeutically relevant heterocycles. Herein, we report the first on-DNA synthesis of multisubstituted indoles via a cascade reaction of Sonogashira coupling and intramolecular ring closure. Further functionalization by Suzuki coupling at the third position exploits a diverse chemical space. The high fidelity of the method also enabled the construction of an indole-based mock library.

N6-Methyladenine Progressively Accumulates in Mitochondrial DNA during Aging.

N6-methyladenine (6mA) in the DNA is a conserved epigenetic mark with various cellular, physiological and developmental functions. Although the presence of 6mA was discovered a few years ago in the nuclear genome of distantly related animal taxa and just recently in mammalian mitochondrial DNA (mtDNA), accumulating evidence at present seriously questions the presence of N6-adenine methylation in these genetic systems, attributing it to methodological errors. In this paper, we present a reliable, PCR-based method to determine accurately the relative 6mA levels in the mtDNA of Caenorhabditis elegans, Drosophila melanogaster and dogs, and show that these levels gradually increase with age. Furthermore, daf-2(-)-mutant worms, which are defective for insulin/IGF-1 (insulin-like growth factor) signaling and live twice as long as the wild type, display a half rate at which 6mA progressively accumulates in the mtDNA as compared to normal values. Together, these results suggest a fundamental role for mtDNA N6-adenine methylation in aging and reveal an efficient diagnostic technique to determine age using DNA.

Cross-Linked α-Synuclein as Inhibitor of Amyloid Formation.

The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.

Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors.

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.

Cross-Linking Mass Spectrometry on P-Glycoprotein.

The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp's conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp's solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskeleton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins.

Optimum collision energies for proteomics: The impact of ion mobility separation.

Ion mobility spectrometry (IMS) is a widespread separation technique used in various research fields. It can be coupled to liquid chromatography-mass spectrometry (LC-MS/MS) methods providing an additional separation dimension. During IMS, ions are subjected to multiple collisions with buffer gas, which may cause significant ion heating. The present project addresses this phenomenon from the bottom-up proteomics point of view. We performed LC-MS/MS measurements on a cyclic ion mobility mass spectrometer with varied collision energy (CE) settings both with and without IMS. We investigated the CE dependence of identification score, using Byonic search engine, for more than 1000 tryptic peptides from HeLa digest standard. We determined the optimal CE values-giving the highest identification score-for both setups (i.e., with and without IMS). Results show that lower CE is advantageous when IMS separation is applied, by 6.3 V on average. This value belongs to the one-cycle separation configuration, and multiple cycles may supposedly have even larger impact. The effect of IMS is also reflected in the trends of optimal CE values versus m/z functions. The parameters suggested by the manufacturer were found to be almost optimal for the setup without IMS; on the other hand, they are obviously too high with IMS. Practical consideration on setting up a mass spectrometric platform hyphenated to IMS is also presented. Furthermore, the two CID (collision induced dissociation) fragmentation cells of the instrument-located before and after the IMS cell-were also compared, and we found that CE adjustment is needed when the trap cell is used for activation instead of the transfer cell. Data have been deposited in the MassIVE repository (MSV000090944).

Collision-Induced Dissociation of Citrullinated Peptide Anions.

Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups reported the usefulness of negative electrospray ionization (ES-) for gaining complementary structural information on peptides and their post-translational modifications (PTM) compared to ES+. Fragmentation of citrullinated peptides has not been previously explored in ES-. In this study, 9 peptides containing citrulline residues were investigated in ES- by stepwise collision energy-dependent measurements on a QTOF instrument and a Q-Orbitrap instrument. Our results of high resolution and mass accuracy show the favored citrulline-selective loss of HNCO from these peptide precursors and their fragments─similarly to that in ES+─along with y-NH3/z, c, c-NH3/b sequence ions. Loss of HNCO from citrullinated peptides in ES- and a proposed mechanism for the reaction have been described here for the first time. HNCO loss intensities from precursors were generally even higher than that in ES+. Interestingly, the most intense fragments corresponded to neutral losses from sequence ions while intact sequence ions were usually minor components of the spectra. High-intensity ions related to cleavages N-terminal to Asp and Glu residues that have been previously reported were also observed. On the other hand, a relatively high number of peaks were observed, possibly due to internal fragmentation and/or scrambling events. While (ES-) MS/MS spectra always require manual inspection and the annotation may be ambiguous, the favorable loss of HNCO and the preferable cleavage N-terminal to Asp residues can be used to differentiate between citrullinated/deamidated sequences.

Compositional Analysis of Glycosaminoglycans in Different Lung Cancer Types-A Pilot Study.

Lung cancer is one of the most commonly diagnosed cancer types. Studying the molecular changes that occur in lung cancer is important to understand tumor formation and identify new therapeutic targets and early markers of the disease to decrease mortality. Glycosaminoglycan chains play important roles in various signaling events in the tumor microenvironment. Therefore, we have determined the quantity and sulfation characteristics of chondroitin sulfate and heparan sulfate in formalin-fixed paraffin-embedded human lung tissue samples belonging to different lung cancer types as well as tumor adjacent normal areas. Glycosaminoglycan disaccharide analysis was performed using HPLC-MS following on-surface lyase digestion. Significant changes were identified predominantly in the case of chondroitin sulfate; for example, the total amount was higher in tumor tissue compared to the adjacent normal tissue. We also observed differences in the degree of sulfation and relative proportions of individual chondroitin sulfate disaccharides between lung cancer types and adjacent normal tissue. Furthermore, the differences in the 6-O-/4-O-sulfation ratio of chondroitin sulfate were different between the lung cancer types. Our pilot study revealed that further investigation of the role of chondroitin sulfate chains and enzymes involved in their biosynthesis is an important aspect of lung cancer research.

Inter- and intratumoral proteomics and glycosaminoglycan characterization of ALK rearranged lung adenocarcinoma tissues: a pilot study.

Lung cancer is one of the most common types of cancer with limited therapeutic options, therefore a detailed understanding of the underlying molecular changes is of utmost importance. In this pilot study, we investigated the proteomic and glycosaminoglycan (GAG) profile of ALK rearranged lung tumor tissue regions based on the morphological classification, mucin and stromal content. Principal component analysis and hierarchical clustering revealed that both the proteomic and GAG-omic profiles are highly dependent on mucin content and to a lesser extent on morphology. We found that differentially expressed proteins between morphologically different tumor types are primarily involved in the regulation of protein synthesis, whereas those between adjacent normal and different tumor regions take part in several other biological processes (e.g. extracellular matrix organization, oxidation-reduction processes, protein folding) as well. The total amount and the sulfation profile of heparan sulfate and chondroitin sulfate showed small differences based on morphology and larger differences based on mucin content of the tumor, while an increase was observed in both the total amount and the average rate of sulfation in tumors compared to adjacent normal regions.

Activation-Free Sulfonyl Fluoride Probes for Fragment Screening.

SuFEx chemistry is based on the unique reactivity of the sulfonyl fluoride group with a range of nucleophiles. Accordingly, sulfonyl fluorides label multiple nucleophilic amino acid residues, making these reagents popular in both chemical biology and medicinal chemistry applications. The reactivity of sulfonyl fluorides nominates this warhead chemotype as a candidate for an external, activation-free general labelling tag. Here, we report the synthesis and characterization of a small sulfonyl fluoride library that yielded the 3-carboxybenzenesulfonyl fluoride warhead for tagging tractable targets at nucleophilic residues. Based on these results, we propose that coupling diverse fragments to this warhead would result in a library of sulfonyl fluoride bits (SuFBits), available for screening against protein targets. SuFBits will label the target if it binds to the core fragment, which facilitates the identification of weak fragments by mass spectrometry.

Protein Biocargo and Anti-Inflammatory Effect of Tomato Fruit-Derived Nanovesicles Separated by Density Gradient Ultracentrifugation and Loaded with Curcumin.

Plant-derived nanovesicles (PDNVs) have become attractive alternatives to mammalian cell-derived extracellular vesicles (EVs) both as therapeutic approaches and drug-delivery vehicles. In this study, we isolated tomato fruit-derived NVs and separated them by the iodixanol density gradient ultracentrifugation (DGUC) into twelve fractions. Three visible bands were observed at densities 1.064 ± 0.007 g/mL, 1.103 ± 0.006 g/mL and 1.122 ± 0.012 g/mL. Crude tomato PDNVs and DGUC fractions were characterized by particle size-distribution, concentration, lipid and protein contents as well as protein composition using mass spectrometry-based proteomics. Cytotoxicity and anti-inflammatory activity of the DGUC fractions associated to these bands were assessed in the lipopolysaccharide (LPS)-stimulated human monocytic THP-1 cell culture. The middle and the low-density visible DGUC fractions of tomato PDNVs showed a significant reduction in LPS-induced inflammatory IL-1ß cytokine mRNA production. Functional analysis of proteins identified in these fractions reveals the presence of 14-3-3 proteins, endoplasmic reticulum luminal binding proteins and GTP binding proteins associated to gene ontology (GO) term GO:0050794 and the regulation of several cellular processes including inflammation. The most abundant middle-density DGUC fraction was loaded with curcumin using direct loading, sonication and extrusion methods and anti-inflammatory activity was compared. The highest entrapment efficiency and drug loading capacity was obtained by direct loading. Curcumin loaded by sonication increased the basal anti-inflammatory activity of tomato PDNVs.

Collision energies: Optimization strategies for bottom-up proteomics.

Mass-spectrometry coupled to liquid chromatography is an indispensable tool in the field of proteomics. In the last decades, more and more complex and diverse biochemical and biomedical questions have arisen. Problems to be solved involve protein identification, quantitative analysis, screening of low abundance modifications, handling matrix effect, and concentrations differing by orders of magnitude. This led the development of more tailored protocols and problem centered proteomics workflows, including advanced choice of experimental parameters. In the most widespread bottom-up approach, the choice of collision energy in tandem mass spectrometric experiments has outstanding role. This review presents the collision energy optimization strategies in the field of proteomics which can help fully exploit the potential of MS based proteomics techniques. A systematic collection of use case studies is then presented to serve as a starting point for related further scientific work. Finally, this article discusses the issue of comparing results from different studies or obtained on different instruments, and it gives some hints on methodology transfer between laboratories based on measurement of reference species.

A carbapenem antibiotic inhibiting a mammalian serine protease: structure of the acylaminoacyl peptidase-meropenem complex.

The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem antibiotic. AAP is a modulator of the ubiquitin-proteasome degradation system and the site of a drug-drug interaction between the widely used antipsychotic, valproate and carbapenems. The active form of pAAP - a toroidal tetramer - binds four meropenem molecules covalently linked to the catalytic Ser587 of the serine-protease triad, in an acyl-enzyme state. AAP is hindered from fully processing the antibiotic by the displacement and protonation of His707 of the catalytic triad. We show that AAP is made susceptible to the association by its unusually sheltered active pockets and flexible catalytic triads, while the carbapenems possess sufficiently small substituents on their ß-lactam rings to fit into the shallow substrate-specificity pocket of the enzyme.

Glycosaminoglycan Analysis of FFPE Tissues from Prostate Cancer and Benign Prostate Hyperplasia Patients Reveals Altered Regulatory Functions and Independent Markers for Survival.

Prostate cancer is one of the most frequent cancer types among men. Several biomarkers and risk assessment methods are already available; however, enhancing their selectivity and sensitivity is still necessary. For improving therapeutic decisions, both basic and clinical research studies are still ongoing for a better understanding of the underlying molecular mechanisms. The enzymatic digests of heparan sulfate (HS) and chondroitin sulfate (CS) chains were investigated in tissue samples taken from patients with prostate cancer (PCa) and benign prostate hyperplasia (BPH) with the HPLC-MS methodology. None of the HS species analyzed showed correlating alterations with currently used markers such as clinical stage, Gleason score, or prostate-specific antigen (PSA) level. The total quantity and sulfation motifs of CS were both significantly different among BPH and different risk groups of PCa. Furthermore, the cancer-specific survival of patients can be predicted based on the levels of non-sulfated and doubly sulfated CS disaccharides as well as the total HS content and the doubly and triply sulfated HS disaccharide ratios. These disaccharide ratios proved to be independent markers from clinical parameters. Further investigations of glycosaminoglycan motifs were proposed for the validation of the results on independent patient cohorts as well.

Arg236 in human chymotrypsin B2 (CTRB2) is a key determinant of high enzyme activity, trypsinogen degradation capacity, and protection against pancreatitis.

Pancreatic chymotrypsins (CTRs) are digestive proteases that in humans include CTRB1, CTRB2, CTRC, and CTRL. The highly similar CTRB1 and CTRB2 are the products of gene duplication. A common inversion at the CTRB1-CTRB2 locus reverses the expression ratio of these isoforms in favor of CTRB2. Carriers of the inversion allele are protected against the inflammatory disorder pancreatitis presumably via their increased capacity for CTRB2-mediated degradation of harmful trypsinogen. To reveal the protective molecular determinants of CTRB2, we compared enzymatic properties of CTRB1, CTRB2, and bovine CTRA (bCTRA). By evolving substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) inhibitory loop variants against the chymotrypsins, we found that the substrate binding groove of the three enzymes had overlapping specificities. Based on the selected sequences, we produced eight SGPI-2 variants. Remarkably, CTRB2 and bCTRA bound these inhibitors with significantly higher affinity than CTRB1. Moreover, digestion of peptide substrates, beta casein, and human anionic trypsinogen unequivocally confirmed that CTRB2 is a generally better enzyme than CTRB1 while the potency of bCTRA lies between those of the human isoforms. Unexpectedly, mutation D236R alone converted CTRB1 to a CTRB2-like high activity protease. Modeling indicated that in CTRB1 Met210 partially obstructed the substrate binding groove, which was relieved by the D236R mutation. Taken together, we identify CTRB2 Arg236 as a key positive determinant, while CTRB1 Asp236 as a negative determinant for chymotrypsin activity. These findings strongly support the concept that in carriers of the CTRB1-CTRB2 inversion allele, the superior trypsinogen degradation capacity of CTRB2 protects against pancreatitis.

Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins.

Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.

Stepwise Collision Energy-Resolved Tandem Mass Spectrometric Experiments for the Improved Identification of Citrullinated Peptides.

The use of tandem mass spectrometry (MS/MS) is a fundamental prerequisite of reliable protein identification and quantification in mass-spectrometry-based proteomics. In bottom-up and middle-down proteomics, proteins are identified by the characteristic fragments of their constituting peptides. Post-translational modifications (PTMs) often further complicate proteome analyses. Citrullination is an increasingly studied PTM converting arginines to citrullines (Cit, X) and is implicated in several autoimmune and neurological diseases as well as different types of cancer. Confirmation of citrullination is known to be very challenging since it results in the same molecular mass change as Asn/Gln deamidation. In this study, we explore which MS/MS characteristics can be used for the reliable identification of citrullination. We synthesized several peptides incorporating Cit residues that model enzymatic cleavages of different proteins with verified or putative citrullination. Collision-induced dissociation was used to investigate the energy dependence of Byonic and Mascot scores and confirmed sequence coverage (CSC) along with the neutral loss of HNCO characteristic to citrulline side chains. We found that although the recommended values (19-45 V) for ramped collision energy settings cover the optimal Mascot, Byonic, or %CSC scores effectively, the diagnostic HNCO loss from precursors and fragments may reach their maximum intensities at lower and higher collision energies, respectively. Therefore, we suggest broadening the ramp range to ∼5-60 V to obtain more favorable identification rates for citrullinated peptides. We also found that Byonic was more successful in correctly identifying citrullinated peptides with deamidated residues than Mascot.

Directed Evolution-Driven Increase of Structural Plasticity Is a Prerequisite for Binding the Complement Lectin Pathway Blocking MASP-Inhibitor Peptides.

MASP-1 and MASP-2 are key activator proteases of the complement lectin pathway. The first specific mannose-binding lectin-associated serine protease (MASP) inhibitors had been developed from the 14-amino-acid sunflower trypsin inhibitor (SFTI) peptide by phage display, yielding SFTI-based MASP inhibitors, SFMIs. Here, we present the crystal structure of the MASP-1/SFMI1 complex that we analyzed in comparison to other existing MASP-1/2 structures. Rigidified backbone structure has long been accepted as a structural prerequisite for peptide inhibitors of proteases. We found that a hydrophobic cluster organized around the P2 Thr residue is essential for the structural stability of wild-type SFTI. We also found that the same P2 Thr prevents binding of the rigid SFTI-like peptides to the substrate-binding cleft of both MASPs as the cleft is partially blocked by large gatekeeper enzyme loops. Directed evolution removed this obstacle by replacing the P2 Thr with a Ser, providing the SFMIs with high-degree structural plasticity, which proved to be essential for MASP inhibition. To gain more insight into the structural criteria for SFMI-based MASP-2 inhibition, we systematically modified MASP-2-specific SFMI2 by capping its two termini and by replacing its disulfide bridge with varying length thioether linkers. By doing so, we also aimed to generate a versatile scaffold that is resistant to reducing environment and has increased stability in exopeptidase-containing biological environments. We found that the reduction-resistant disulfide-substituted l-2,3-diaminopropionic acid (Dap) variant possessed near-native potency. As MASP-2 is involved in the life-threatening thrombosis in COVID-19 patients, our synthetic, selective MASP-2 inhibitors could be relevant coronavirus drug candidates.

Expression of glycosaminoglycans in cirrhotic liver and hepatocellular carcinoma-a pilot study including etiology.

Chronic liver diseases have both high incidence and mortality rates; therefore, a deeper understanding of the underlying molecular mechanisms is essential. We have determined the content and sulfation pattern of chondroitin sulfate (CS) and heparan sulfate (HS) in human hepatocellular carcinoma and cirrhotic liver tissues, considering the etiology of the diseases. A variety of pathological conditions such as alcoholic liver disease, hepatitis B and C virus infections, and primary sclerosing cholangitis were studied. Major differences were observed in the total abundance and sulfation pattern of CS and HS chains. For example, the 6-O-sulfation of CS is fundamentally different regarding etiologies of cirrhosis, and a 2-threefold increase in HS N-sulfation/O-sulfation ratio was observed in hepatocellular carcinoma compared to cirrhotic tissues.