V8 Protease Research Articles

Mechanisms and Therapeutic Targets of Staphylococcus Aureus - Induced Itch in Atopic Dermatitis

Atopic dermatitis is a chronic skin disorder marked by inflammation, erythema, and pruritus, often beginning in childhood and characterized by exacerbations and remissions. Its etiology involves genetic, immunological, and environmental factors. Staphylococcus aureus, a common pathogen, exacerbates atopic dermatitis by producing toxins and enzymes such as the serine protease V8, which activates the protease-activated receptor 1 (PAR1) on sensory neurons, inducing itch. This mechanism highlights potential therapeutic targets for alleviating pruritus. Research into the physiological pathways of itch, including the role of PAR1 and other protease-activated receptors, reveals promising strategies for treatment. PAR1 antagonists could be repurposed to treat chronic itch, providing new therapeutic avenues. PAR1 and other protease-activated receptors, is crucial in developing new treatment strategies. Anti-pruritic therapies targeting these pathways, such as PAR1 antagonists, show promise in mitigating itch symptoms. Moreover, existing drugs that inhibit PAR1 could be repurposed for treating chronic itch, providing a new avenue for relief in patients with atopic dermatitis. Additionally, the evolutionary role of itch induced by microorganisms suggests pathogens may exploit neural reflexes to enhance their spread. Advancements in understanding the mechanisms behind S. aureus-induced itch and the physiological pathways involved offer promising new directions for therapeutic intervention. Advancements in understanding the mechanisms of Staphylococcal aureus-induced itch and associated physiological pathways offer promising directions for therapeutic intervention, potentially improving management and treatment outcomes for patients with atopic dermatitis and other pruritic conditions.

Staphylococcus aureus: The Bug Behind the Itch in Atopic Dermatitis

Pruritus or itch is a defining symptom of atopic dermatitis (AD). The origins of itch are complex, and it is considered both a defense mechanism and a cause of disease that leads to inflammation and psychological stress. Considerable progress has been made in understanding the processes that trigger itch, particularly the pruritoceptive origins that are generated in the skin. This perspective review discusses the implications of a recent observation that the V8 protease expressed by Staphylococcus aureus can directly trigger sensory neurons in the skin through activation of protease-activated receptor 1. This may be a key to understanding why itch is so common in AD because S. aureus commonly overgrows in this disease owing to deficient antimicrobial defense from both the epidermis and the cutaneous microbiome. Increased understanding of the role of microbes in AD provides increased opportunities for safely improving the treatment of this disorder.

Better than being aPARt: S. aureus itches to get close to sensory neurons

In a recent issue of Cell, Deng etal. show that S.aureus serine protease V8 triggers itch, independent of inflammation, by activating sensory neurons through PAR1. This study presents mechanistic insights into pruritogenic bacteria and their interactions with sensory neurons while providing a possible approach for treating itch-related diseases.

Staphylococcus scratches its itch

Itch exacerbates infection and inflammation-associated skin pathology. In this issue of Cell, Deng etal. identify a V8 protease released by Staphylococcus aureus triggering itch via neuronal protease-activated receptor 1. In so doing, they uncover profound consequences of microbial neurosensory modulation and the ensuing scratch-induced tissue damage that potentiates infection.

S. aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to microbes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruriceptor sensory neurons to drive itch. Epicutaneous S.aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S.aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-activated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S.aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch.

Necrotic activity of ExhC from Mammaliicoccus sciuri is mediated by specific amino acid residues

Mammaliicoccus sciuri, a commensal and pathogenic bacterium of significant clinical and veterinary relevance, expresses exfoliative toxin C (ExhC), a specific glutamyl endopeptidase belonging to the chymotrypsin family as the principal virulence factor. However, unlike most members of this family, ETs are inactive against a wide range of substrates and possess exquisite specificity for desmoglein-1 (Dsg1), a cadherin-like adhesion molecule that is crucial to maintain tissue integrity, thereby preventing the separation of skin cells and the entry of pathogens. ExhC is of clinical importance since in addition to causing exfoliation in pigs and mice, it induces necrosis in multiple mammalian cell lines, a property not observed for other ETs. Previous experiments have implicated the ExhC79–128 fragment in causing necrosis. Site-directed mutagenesis of specific residues within this fragment were studied and led to the design of an ExhC variant containing four-point mutations (ExhCmut4) lacking necrotic potential but retaining nearly wild-type (wt) levels of enzymatic activity. Moreover, the determination of the ExhCwt and ExhCmut4 crystal structures identified the conformation in the necrosis-linked region. These results constitute an important step toward the understanding of the mechanisms underlying the necrotic and epidermolytic activity of ExhC.

A low molecular weight fraction of wheat bran aqueous extract acts as a promoter of fused nanospheres after proteolysis with V8

Protein-rich wheat bran aqueous extract was fractionated by size exclusion chromatography. Each of the chromatographic fractions was hydrolyzed with V8 protease in a reaction medium containing Ca2+. Both the fractions and the hydrolysates were characterized physically and chemically. The objective was to obtain information about which components are involved in the formation of nanoparticles, reported in previous works. Six chromatographic fractions from 0.3 to 102.8 kDa were obtained. The results of chemical characterization, electrophoresis, UV-Vis spectroscopy, and infrared spectroscopy revealed the presence of protein-polysaccharide complexes in four of the fractions. Fused nanospheres at different angles were found by scanning electron microscopy after proteolysis of a 15.1 kDa fraction. It was proposed that such binding could provide sites to form branched nanostructures.

Diflunisal Attenuates Virulence Factor Gene Regulation and Phenotypes in Staphylococcus aureus.

Virulence factor expression is integral to pathogenicity of Staphylococcus aureus. We previously demonstrated that aspirin, through its major metabolite, salicylic acid (SAL), modulates S. aureus virulence phenotypes in vitro and in vivo. We compared salicylate metabolites and a structural analogue for their ability to modulate S. aureus virulence factor expression and phenotypes: (i) acetylsalicylic acid (ASA, aspirin); (ii) ASA metabolites, salicylic acid (SAL), gentisic acid (GTA) and salicyluric acid (SUA); or (iii) diflunisal (DIF), a SAL structural analogue. None of these compounds altered the growth rate of any strain tested. ASA and its metabolites SAL, GTA and SUA moderately impaired hemolysis and proteolysis phenotypes in multiple S. aureus strain backgrounds and their respective deletion mutants. Only DIF significantly inhibited these virulence phenotypes in all strains. The kinetic profiles of ASA, SAL or DIF on expression of hla (alpha hemolysin), sspA (V8 protease) and their regulators (sigB, sarA, agr (RNAIII)) were assessed in two prototypic strain backgrounds: SH1000 (methicillin-sensitive S. aureus; MSSA) and LAC-USA300 (methicillin-resistant S. aureus; MRSA). DIF induced sigB expression which is coincident with the significant inhibition of RNAIII expression in both strains and precedes significant reductions in hla and sspA expression. The inhibited expression of these genes within 2 h resulted in the durable suppression of hemolysis and proteolysis phenotypes. These results indicate that DIF modulates the expression of key virulence factors in S. aureus via a coordinated impact on their relevant regulons and target effector genes. This strategy may hold opportunities to develop novel antivirulence strategies to address the ongoing challenge of antibiotic-resistant S. aureus.

Antimicrobial Effect of Nitric Oxide Releasing Hydrogels on Staphylococcus Aureus Derived Proteases

AbstractThe skin serves as a crucial barrier against environmental insults and invading pathogens. However, traumatic injury or skin disorder can compromise this barrier function, leasing to bacterial colonization and infection of the wound with microorganisms such as Staphylococcus aureus, which are normally present on healthy skin. The secretion of bacterial proteases such as V8 protease from S. aureus can disturb the equilibrium between extracellular matrix degradation and deposition during wound healing resulting in loss of barrier integrity. We report the feasibility of a nitric oxide (NO) releasing poly‐ε‐lysine (pεK) hydrogel to prevent loss of barrier function caused by V8 proteases. The fabrication and characterization of the pεK hydrogel and NO releasing properties in biologically relevant media are reported. The NO‐releasing pεK hydrogel have demonstrated bactericidal activity against a clinical isolate of S. aureus in complex physiological media and concurrently reduce the catalytic activity of secreted V8 protease. Moreover, pεK hydrogels are cytocompatible with keratinocytes and dermal fibroblasts. In contrast, Penicillin G loaded pεK hydrogels showed excellent antimicrobial efficacy but did not affect V8 catalytic activity. This demonstrates that NO‐releasing pεK hydrogels hold potential as an effective treatment for infected wounds reducing the microbial burden and inactivating bacterial secreted proteases.

Discovery and validation of glucose-sensitive peptide biomarkers from human serum albumin to diagnose type 2 diabetes mellitus

Glycated albumin (GA), which represents the global glycation level of albumin, has emerged as a biomarker for diagnosing prediabetes and diabetes. In our previous study, we developed a peptide-based strategy and found three putative peptide biomarkers from the tryptic peptides of GA to diagnose type 2 diabetes mellitus (T2DM). However, the trypsin cleavage sites at the carboxyl side of lysine (K) and arginine (R) are consistent with the nonenzymatic glycation modification site residues, which considerably increases the number of missed cleavage sites and half-cleaved peptides. To solve this problem, the endoproteinase Glu-C was used to digest GA from human serum to screen putative peptides to diagnose T2DM. In the discovery phase, we found eighteen and fifteen glucose-sensitive peptides from purified albumin and human serum incubated with 13C glucose in vitro, respectively. In the validation phase, eight glucose-sensitive peptides were screened and validated in 72 clinical samples (28 healthy controls and 44 patients with diabetes) using label-free LC-ESI-MRM. Three putative sensitive peptides (VAHRFKDLGEE, FKPLVEEPQNLIKQNCE and NQDSISSKLKE) from albumin exhibited good specificity and sensitivity based on receiver operating characteristic analysis. In summary, three peptides were found as promising biomarkers for the diagnosis and assessment of T2DM based on mass spectrometry.

Targeted Bottom-Up Mass Spectrometry Approach for the Relative Quantification of Post-Translational Modification of Bovine κ-Casein during Milk Fermentation.

κ-casein (κ-CN) is one of the key components in bovine milk, playing a unique role in the structuration of casein micelles. It contains in its chemical structure up to sixteen amino acid residues (mainly serine and threonine) susceptible to modifications, including glycosylation and phosphorylation, which may further be formed during milk processing. In this study, changes in post-translational modification (PTM) of κ-CN during bovine milk fermentation were investigated. One-to-five-day fermented milk samples were produced. A traditional bottom–up proteomics approach was used to establish a multiple-reaction monitoring (MRM) method for relative quantification of κ-CN PTM. Endoproteinase Glu-C was found to efficiently digest the κ-CN molecule. The developed LC-MS method was validated by performing assessments of linearity, precision, repeatability, reproducibility, limit of detection (LOD), and limit of quantification (LOQ). Among the yielded peptides, four of them containing serine and threonine residues were identified and the unmodified as well as the modified variants of each of them were relatively quantified. These peptides were (1) IPTINTIASGEPTSTTE [140, 158], (2) STVATLE [162, 168], (3) DSPE [169, 172], and (4) INTVQVTSTAV [180, 190]. Distribution analysis between unmodified and modified peptides revealed that over 50% of κ-CN was found in one of its modified forms in milk. The fermentation process further significantly altered the composition between unmodified/modified κ-CN, with glycoslaytion being predominant compared to phosphorylation (p < 0.01). Further method development towards α and β-CN fractions and their PTM behavior would be an asset to better understand the changes undergone by milk proteins and the micellar structure during fermentation.

Construction of an attenuated glutamyl endopeptidase deletion strain of Nocardia seriolae

The glutamyl endopeptidase homolog of Nocardia seriolae (GluNS) has been proved to be a potential virulence factor in our previous study. Present investigation was carried out to construct an attenuated N. seriolae strain by deletion with GluNS gene and evaluate its protective immunity in head snakehead. A deletion strain (NS-ΔGluNS) was established by knockout of gene GluNS from wild strain N. seriolae ZJ0503 via homologous recombination. The LD50 of NS-ΔGluNS in 3.41 × 106 cfu/mL was significantly increased than that of wild strain in 4.75 × 105 cfu/mL, indicating that the virulence of N. seriolae has been attenuated with the knockout of GluNS. Meanwhile, applying NS-ΔGluNS as an attenuated live vaccine to immune hybrid snakehead, the non-specific immunity parameters (serum LYZ, POD, ACP, and AKP activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, CD4, and IL-8) expression were up-regulated in different tissues, which indicated that they were able to trigger humoral and cell-mediated immune responses. Furthermore, the protective efficacy in hybrid snakehead after vaccination with NS-ΔGluNS shown 73.53% relative percentage survival (RPS). Taken together, the attenuated NS-ΔGluNS was obtained successfully and it could elicit strong immune response and supply protective efficacy to hybrid snakehead against N. seriolae wild strain.

Multiplexed quantification of insulin and C-peptide by LC-MS/MS without the use of antibodies

IntroductionThe measurement of insulin and C-peptide provides a valuable tool for the clinical evaluation of hypoglycemia. In research, these biomarkers are used together to better understand hyperinsulinemia, hepatic insulin clearance, and beta cell function. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an attractive approach for the analysis of insulin and C-peptide because the platform is specific, can avoid certain limitations of immunoassays, and can be multiplexed. Previously described LC-MS/MS methods for the simultaneous quantification of insulin and C-peptide measure the intact analytes and most have relied on immunoaffinity enrichment. These approaches can be limited in terms of sensitivity and interference from auto-antibodies, respectively. We have developed a novel method that does not require antibodies and uses proteolytic digestion to yield readily ionizable proteotypic peptides that enables the sensitive, specific, and simultaneous quantitation of insulin and C-peptide. MethodsSerum samples were precipitated with acetonitrile. Analytes were enriched using solid phase extraction and then digested with endoproteinase Glu-C. Surrogate peptides for insulin and C-peptide were analyzed using targeted LC-MS/MS. ResultsInter-day imprecision was below 20 %CV and linearity was observed down to the lower limit of quantitation for both analytes (insulin = 0.09 ng/mL, C-peptide = 0.06 ng/mL). Comparison to a commercially available insulin immunoassay (Beckman Coulter UniCel DxI 600 Access) revealed a 30% bias between methods. ConclusionA novel LC-MS/MS method for the simultaneous analysis of insulin and C-peptide using Glu-C digestion was developed and evaluated. A detailed standard operating procedure is provided to help facilitate implementation in other laboratories.

A proteomics strategy for the identification of multiple sites in sulfur mustard–modified HSA and screening potential biomarkers for retrospective analysis of exposed human plasma

A major challenge for the unequivocal verification of alleged exposure to sulfur mustard (HD) lies in identifying its multiple modifications on endogenous proteins and utilizing these modified proteins to achieve accurate, sensitive, and rapid detection for retrospective analysis of HD exposure. As the most abundant protein in human plasma, human serum albumin (HSA) can react with many xenobiotics, such as HD, to protect the body from damage. The HSA adducts induced by HD have been used as biomarkers for the verification of HD exposure. In this study, the modification sites on HSA by HD were identified through application of the bottom-up strategy used in proteomics, and 41 modified sites were discovered with seven types of amino acids, of which 3 types were not previously reported. Then, different enzymes, including pepsin, endoproteinase Glu-C, and pronase, were applied to digest HD-HSA to produce adducts with hydroxyethylthioethyl (HETE) groups, which may be used as potential biomarkers for HD exposure. As candidates for retrospective analysis, sixteen adducts were obtained and characterized with ultra-high-pressure liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (UHPLC-QE Focus MS). These potential biomarkers were evaluated in human plasma that was exposed in vitro to HD and five of its analogues. This study integrated the identification of modification sites through application of the bottom-up strategy of proteomics and screening biomarkers, providing a novel strategy for retrospective detection of the exposure of xenobiotic chemicals.

Development and certification of a pharmacopoeial reference standard for primary structure identification of purified recombinant interferon beta-1b by peptide mapping

Medicines based on recombinant human interferons (rhIFNs) beta-1a and beta-1b are used as first-line treatment of multiple sclerosis. Meanwhile, rhIFN beta-1a and beta-1b have structural differences associated with the eukaryotic or prokaryotic expression systems, respectively. Pharmacopoeias require identification of the primary structure of recombinant proteins by peptide mapping, which involves the use of reference material. Currently, there is no international reference standard available for rhIFN beta-1b structural identification. The aim of the study was development and certification of a pharmacopoeial reference standard for identification of the amino acid sequence of purified rhIFN beta-1b by peptide mapping. Materials and methods: rhIFN beta-1b produced by GENERIUM and endoproteinase Glu-C from Staphylococcus aureus V8 were used in the study. The peptide mapping was performed using reverse-phase high-performance liquid chromatography (RP HPLC) and high-resolution mass spectrometry. Statistical evaluation of the results included calculation of the arithmetic mean, standard deviation, and coefficient of variation. Results: the authors developed and certified a Russian Pharmacopoeia reference standard for structural identification of rhIFN beta-1b (PhRS 3.2.00447). The certified characteristic is the range of retention times of characteristic peaks: the absolute retention time was 42.0–43.2 for the third (reference) peak, the relative retention time was 0.61–0.66 for the first peak, 0.68–0.73 for the second peak, 1.04–1.06 for the fourth peak, 1.14–1.15 for the fifth peak, 1.22–1.24 for the sixth peak, and 1.29–1.30 for the seventh peak. Conclusions: the authors developed requirements for the rhIFN beta-1b pharmacopoeial reference standard. The material chosen as the candidate reference standard was an intermediate rhIFN beta-1b product sampled before addition of human serum albumin. The quality control was carried out in accordance with the developed specification. The authors analysed the amino acid sequence of the molecule, confirmed the presence of the disulfi e bond, and obtained the certifi d characteristic of the reference standard. Comparative analysis of the peptide maps of the certified rhIFN beta-1b pharmacopoeial reference standard and the rhIFN beta-1a reference standard revealed differences between the maps, and, therefore, confirmed the relevance of the developed reference standard.

Site-Specific Characterization of Heat-Induced Disulfide Rearrangement in Beta-Lactoglobulin by Liquid Chromatography-Mass Spectrometry.

Disulfides are important for maintaining the protein native structure, but they may undergo rearrangement in the presence of free Cys residues, especially under elevated temperatures. Disulfide rearrangement may result in protein aggregation, which is associated with in vivo pathologies in organisms and in vitro protein functionality in food systems. In a food context, it is therefore important to understand the process of disulfide rearrangement on a site-specific level in order to control aggregation. In the present study, a liquid chromatography-mass spectrometry (LC-MS)-based bottom-up site-specific proteomic approach was optimized to study disulfide rearrangements in beta-lactoglobulin (β-LG) under different heat treatments (60-90 °C). Artifactual disulfide rearrangement observed during sample preparation using a conventional protocol was detected and minimized by blocking the remaining free Cys residues with iodoacetamide in the presence of urea after heat treatment. Use of endoproteinase Glu-C for enzymatic hydrolysis allowed, for the first time, identification and comparison of the relative intensity of all theoretically possible β-LG disulfide cross-links formed by the heat treatments. Non-native disulfides were formed from heat treatment at approx. 70 °C where β-LG started to unfold, while higher levels of inter-molecular disulfide links were formed at ≥80 °C, in agreement with β-LG aggregation detected by size exclusion chromatography analysis. Collectively, the Cys residues of the surface-located native disulfide Cys66-Cys160 were proposed to be more reactive, participating in heat-induced disulfide rearrangement, compared to other Cys residues. The abundant signal of non-native disulfide bonds containing Cys66, especially Cys66-Cys66, observed after heating suggested that Cys66 is a key disulfide-linked Cys residue in β-LG participating in heat-induced inter-molecular disulfide bonds and the corresponding protein aggregation.

One-Step Isolation of Protein C-Terminal Peptides from V8 Protease-Digested Proteins by Metal Oxide-Based Ligand-Exchange Chromatography

We have developed a one-step method to isolate protein C-terminal peptides from V8 protease-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, affording a digested peptide with two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the nonterminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After the optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 μg of peptides (10 μg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 μg of HeLa peptides was further divided into six fractions by high-pH reversed-phase liquid chromatography (LC) prior to nanoLC/MS/MS, 2203 protein C-termini were identified with less than 3% contamination with internal peptides. We believe that this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive, and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.

Efficacy of Heparinoid Cream Containing Pseudo-Ceramide for Remission of Atopic Dermatitis

PurposeAtopic dermatitis (AD) is characterized by chronic inflammation, which frequently recurs, is exacerbated, and enters remission. A maintenance remission period is important for AD patients. We developed a formulation for use during AD remission, containing heparinoid and pseudo-ceramide that forms a lamellar structure. We evaluated the allergen permeability and examined the formulation’s efficacy in maintaining remission in patients with AD.Materials and MethodsSeventeen AD patients applied a cream containing 0.3% heparinoid and pseudo-ceramide (test cream group, n = 10), or a general cream containing 0.3% heparinoid (control cream group, n = 7) to their arm for four weeks after inducing remission with the application of a steroid cream for two weeks.ResultsThe lamellar structure of the test cream was confirmed with small- and wide-angle x-ray scattering analysis and observation by transmission electron microscopy. The test cream inhibited the penetration of V8 protease significantly compared to the control cream in vitro. According to AD severity score by dermatologists, the effects remission maintenance of the test cream group were comparable to those of the control cream group. However, the test cream group had a significantly increased skin hydration value compared to the control cream group. A significant decrease in transepidermal water loss, an indicator of skin barrier function, was shown in the test cream group compared to the control cream group.ConclusionThe cream with lamellar structures containing heparinoid and pseudo-ceramides may inhibit allergen penetration. Moreover, skin properties improved during the remission period; thus, the formulation we developed was suitable for use during the AD remission period.

Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation

SummaryMitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.

Histone Post‐Translational Modifications Detected in Mozambique Tilapia ( Oreochromis mossambicus ) Using Three Digestion Conditions

To analyze histone post-translational modifications (PTMs) through mass spectrometry, both bottom-up and middle-down approaches to proteomics are commonly employed. In this study, we compared the histone PTMs that can be detected in Mozambique tilapia (Oreochromis mossambicus) using three different digestion conditions. First, we tested a bottom-up approach where histone proteins are propionylated then digested with trypsin for cleavage on the carboxyl end of arginine. Second, we tested a middle-down approach where histones are digested with the V8 protease in the buffer ammonium bicarbonate. This leads to cleavage on the carboxyl end of glutamate. We tested another middle-down approach using V8 in the buffer sodium phosphate for the third digestion condition. This leads to cleavage on the carboxyl end of both glutamate and aspartate. Our results indicate which histone PTMs can be identified under each digestion condition on samples of gills, kidney, and testes of Mozambique tilapia. We specify which PTMs are only detected using one method, and which are detected through multiple methods.