Novel contribution to clubfoot pathogenesis: The possible role of extracellular matrix proteins.

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Identification of short-chain poly-3-hydroxybutyrates in Saiga horn extracts using LC-MS/MS.

Saiga horn extracts were analyzed with the goal of obtaining new information about compounds present in it. The purpose of this study is to find synthetic alternatives to Saiga horn extract, which is used in traditional Chinese medicine, by identifying potentially biologically active compounds in the extracts. Using high-performance liquid chromatography coupled with high-resolution mass spectrometry, we have been able to identify a series of short-chain polyhydroxybutyrates in alcoholic extracts of Saiga horn. Optimized high-performance liquid chromatography coupled with tandem mass spectrometry methods for analysis of short-chain poly-3-hydroxybutyrates were developed and subsequently applied to investigate Saiga horn extract for the presence of these compounds, which might explain its biological actions, particularly for its antipyretic and procoagulant properties.

Proteomic analysis of dentin-enamel junction and adjacent protein-containing enamel matrix layer of healthy human molar teeth.

The dentin-enamel junction (DEJ) is the border where two different mineralized structures - enamel and dentin - meet. The protein-rich DEJ, together with the inner enamel region of mature teeth, is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the DEJ and the adjacent enamel organic matrix (EOM). We studied proteomes of the DEJ and EOM of healthy human molars and compared them with dentin and enamel proteomes from the same teeth. These tissues were cut out of tooth sections by laser capture microdissection, proteins were extracted and cleaved by trypsin, then processed by liquid chromatography coupled to tandem mass spectrometry to analyze the proteome profiles of these tissues. This study identified 46 proteins in DEJ and EOM. The proteins identified have a variety of functions, including calcium ion-binding, formation of extracellular matrix, formation of cytoskeleton, cytoskeletal protein binding, cell adhesion, and transport. Collagens were identified as the most dominant proteins. Tissue-specific proteins, such as ameloblastin and amelogenin, were also detected. Our findings reveal new insight into proteomics of DEJ and EOM, highly mineralized tissues that are obviously difficult to analyze.

Proteomic analysis of cardiac ventricles: baso-apical differences.

The heart is characterized by a remarkable degree of heterogeneity. Since different cardiac pathologies affect different cardiac regions, it is important to understand molecular mechanisms by which these parts respond to pathological stimuli. In addition to already described left ventricular (LV)/right ventricular (RV) and transmural differences, possible baso-apical heterogeneity has to be taken into consideration. The aim of our study has been, therefore, to compare proteomes in the apical and basal parts of the rat RV and LV. Two-dimensional electrophoresis was used for the proteomic analysis. The major result of this study has revealed for the first time significant baso-apical differences in concentration of several proteins, both in the LV and RV. As far as the LV is concerned, five proteins had higher concentration in the apical compared to basal part of the ventricle. Three of them are mitochondrial and belong to the "metabolism and energy pathways" (myofibrillar creatine kinase M-type, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase). Myosin light chain 3 is a contractile protein and HSP60 belongs to heat shock proteins. In the RV, higher concentration in the apical part was observed in two mitochondrial proteins (creatine kinase S-type and proton pumping NADH:ubiquinone oxidoreductase). The described changes were more pronounced in the LV, which is subjected to higher workload. However, in both chambers was the concentration of proteins markedly higher in the apical than that in basal part, which corresponds to the higher energetic demand and contractile activity of these segments of both ventricles.

Proteins and their modifications in a medieval mummy.

Proteins and their modifications of the natural mummy of Cangrande della Scala (Prince of Verona, Northern Italy, 1291-1329) were studied. The nano-LC-Q-TOF analysis of samples of rib bone and muscle from the mummy showed the presence of different proteins including Types I, III, IV, V, and XI collagen, hemoglobin (subunits alpha and beta), ferritin, biglycan, vitronectin, prothrombin, and osteocalcin. The structure of Type I and Type III collagen was deeply studied to evaluate the occurrence of modifications in comparison with Type I and Type III collagen coming from tissues of recently died people. This analysis showed high percentage of asparaginyl and glutaminyl deamidation, carbamylation and carboxymethylation of lysine, as well as oxidation and dioxidation of methionine. The most common reaction during the natural mummification process was oxidation-the majority of lysine and proline of collagen Type I was hydroxylated whereas methionine was oxidated (oxidated or dioxidated). To the best of our knowledge, this is the first study which reports the protein profile of a natural mummified human tissue and the first one which describes the carbamylation and carboxymethylation of lysine in mummified tissues.

Proteomic analysis of human tooth pulp proteomes - Comparison of caries-resistant and caries-susceptible persons.

UNLABELLED: Most people in the world suffer from dental caries, >90% of adults experience caries on enamel and root surfaces during their life. However, the overall roles of all factors in the development of dental caries still remain unclear and are worthy of recent investigation. In this study we used a proteomic 2D-DIGE approach in connection with MS/MS to investigate the different protein abundances in the tooth pulp of human third molars obtained from caries-resistant and caries-susceptible people. Statistical analysis of the two protein maps obtained on large gel (17cm length) and mini gel (7cm length) followed by nLC-MS/MS analysis enabled the identification of 16 significantly changed spots with unique protein identifications corresponding to 12 non-redundant proteins. Seven proteins exhibited higher and four proteins exhibited lower expression in the caries-resistant samples compared to the caries-susceptible samples. Additionally, one protein (alpha-1-antitrypsin) exhibited both expressions (up and down). Most of the differentially expressed proteins were associated with protein metabolism, energy production, cytoskeletal organization and transport. These differentially expressed proteins are likely involved in the natural resistance or susceptibility of humans to the development of dental caries and suggest that the resistance mechanism is multifactorial. BIOLOGICAL SIGNIFICANCE: Dental caries are not a serious and life-threatening disease, but their healing requires many remedies and takes up a lot of time. Moreover, neglecting the problem may lead to tooth loss, which can strongly reduce the quality of life. Therefore the identifying effective and safe oral medicine and determining the causes of caries-resistance were viewed as the main aims of this study. Our work aims to elucidate the mechanism of natural human resistance to the development of dental caries by studying the proteomes of tooth pulp isolated from patients who displayed different prevalences of tooth caries. This study is the first protein tooth pulp comparison of sound teeth obtained from caries-resistant versus caries-susceptible people.

Proteomic analysis of human tooth pulp: proteomics of human tooth.

INTRODUCTION: The unique pulp-dentin complex demonstrates strong regenerative potential, which enables it to respond to disease and traumatic injury. Identifying the proteins of the pulp-dentin complex is crucial to understanding the mechanisms of regeneration, tissue calcification, defense processes, and the reparation of dentin by dental pulp. The lack of knowledge of these proteins limits the development of more efficient therapies. METHODS: The proteomic profile of human tooth pulp was investigated and compared with the proteome of human dentin and blood. The samples of tooth pulp were obtained from 5 sound permanent human third molars of 5 adults (n = 5). The extracted proteins were separated by 2-dimensional gel electrophoresis, analyzed by nano-liquid chromatography tandem mass spectrometry, and identified by correlating mass spectra to the proteomic databases. RESULTS: A total of 342 proteins were identified with high confidence, and 2 proteins were detected for the first time in an actual human sample. The identified tooth pulp proteins have a variety of functions: structural, catalytic, transporter, protease activity, immune response, and many others. In a comparison with dentin and blood plasma, 140 (pulp/dentin) shared proteins were identified, 37 of which were not observed in plasma. It can be suggested that they might participate in the unique pulp-dentin complex. CONCLUSIONS: This proteomic investigation of human tooth pulp, together with the previously published study of human dentin, is one of the most comprehensive proteome lists of human teeth to date.

Monotopic modifications derived from in vitro glycation of albumin with ribose.

Post-translational modifications are significant reactions that occur to proteins. One of these modifications is a non-enzymatic reaction between the oxo-group(s) of sugars and amino-group(s) of protein - glycation. This reaction plays an important role in the chronic complications of diabetes mellitus, or in the aging process of organisms, that is, it has an important role in the pathophysiology and "normal" physiology of animals. In the work presented here, we studied the glycation of albumins (HSA and BSA). Methodologically, we used nano-LC coupled to a QTOF mass spectrometer. In vitro-modified proteins were cleaved by trypsin and the arising peptides were separated on a C(18) nano column with a trap-column. Peptides and their modifications were analysed with a high-resolution QTOF mass spectrometer with a mass determination precision of better than 5 ppm. Non-enzymatic in vitro reaction products between albumin and ribose were identified. Besides well-known carboxymethyl lysine, new modifications were determined - creating mass shifts of 78 and 218. The origin of the first modification is discussed and its possible structure is presented. In addition, a mass shift of 132 belonging to a Schiff base was also identified. The location of all the modifications within the structure of the proteins was determined and their reactivity to various oxo-compounds was also examined.

Comprehensive proteomic analysis of human dentin.

Proteomic analysis of the human body is a significant recent scientific endeavour. In this study, we investigated the proteomic profile of human dentin using modern analytical and mass spectrometric techniques. Five healthy permanent human molars from five adults were cut, pulverized, denaturated with guanidine buffer, and demineralized with EDTA buffer. The extracted proteins were analysed by gel electrophoresis (SDS-PAGE and two-dimensional gel electrophoresis), digested with trypsin, and separated by liquid chromatography/high-resolution tandem mass spectrometry. We identified 289 proteins with high confidence, 90 of which had not been previously detected in human dentin. Nine (currently hypothetical) proteins were identified for the first time in an actual human sample. The proteins have a variety of functions, including calcium-ion binding, formation of the extracellular matrix, formation of the cytoskeleton, cytoskeletal protein binding, immune response, and transport. In conclusion, this is the first use of two-dimensional electrophoresis for investigating human dentin.

Study of saiga horn using high-performance liquid chromatography with mass spectrometry.

The saiga horns have been investigated the using of modern analytic methods. High-performance liquid chromatography (HPLC) with mass-spectrometric (MS and MS/MS) detection and polyacrylamide gel electrophoresis (PAGE) were used. It could be concluded that basic proteins of the saiga horns are keratins and collagen. The basic representation protein in all samples is keratin type I microfibrillar (from sheep), keratin type II microfibrillar (from sheep), collagen type I (α(1)) (from bovine) and collagen type I (α(2)) (from bovine). Free amino acids we determined in all samples are nontreated by enzyme.

Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by high-performance liquid chromatography-mass spectrometry and capillary zone electrophoresis-mass spectrometry.

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by various oxo-compounds (glucose, ribose, glyoxal and glutardialdehyde) have been investigated using high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). Both of these methods used mass spectrometric (MS) detection. Three enzymes (trypsin, pepsin, proteinase K) were used to digest glycated BSA. The extent of modification depended on the selected oxo-compound. Reactivity increased progressively from glucose to glutardialdehyde (glucose

Determination of insoluble avian eggshell matrix proteins.

The organic components of bones and other mineralized tissues have a high impact on the organization and deposition of calcium, and consequently influence the mechanical properties of those tissues. The extractable proteins of avian eggshells have been studied extensively and many of them have been identified; insoluble (non-extractable) proteins have been sparsely studied, however. In the work discussed in this paper we studied EDTA-insoluble proteins by gradual decalcification of eggshell with EDTA. The insoluble proteinaceous films were chemically treated with cyanogen bromide and the mixtures of large fragments obtained were gradually precipitated with salt. The separated fractions were digested with trypsin and analyzed by HPLC-MS-MS (ion trap mass spectrometer). Analysis of the entire eggshell matrix (without precipitation steps) only enabled 6 proteins to be determined (ovocalyxins 32 and 36, ovocleidin 17 and 116, clusterin, and ovalbumin). Pretreatment of the individual eggshell layers and gradual precipitation with salt markedly increased the number of proteins identified - 28 proteins were determined. We identified for the first time collagens I (two chains) and III in the eggshell matrix, and Kunitz-like protease inhibitor as a major shell matrix protein. Besides the above mentioned proteins we can also mention EDIL3, fibronectin, sulfhydryl oxidase, tubulin alpha 1, lysozyme, Dickkopf-related protein 3, keratins, and ovotransferrin. The relative abundances of proteins in all eggshell layers were determined using the exponentially modified protein abundance index (emPAI). In the cuticle layer seven proteins were identified, whereas 16 proteins were described in the palisade layer and 23 in the mammillary layer.

Separation of tryptic peptides of native and glycated BSA using open-tubular CEC with salophene-lanthanide-Zn(2+) complex as stationary phase.

Open-tubular CEC (OT-CEC) with a new stationary phase, salophene-lanthanide-Zn(2+) complex, has been applied to the separation of tryptic peptides of native BSA and BSA glycated by glucose and ribose. Glycation of proteins (non-enzymatic modification by sugars) significantly affects their properties and it is of great importance from a physiological point of view. Separation of tryptic peptides of glycated BSA by CZE was poor because of their strong adsorption to the bare fused silica capillary. An improved separation of tryptic peptides of both native and glycated BSA was achieved by OT-CEC in the fused silica capillary non-covalently coated with salophene-lanthanide-Zn(2+) complex, which suppressed the adsorption of peptides to the capillary and via specific interactions with some (glyco)peptides enhanced selectivity of the separation. Significant differences have been found in OT-CEC analyses of tryptic hydrolysates of native and glycated BSA. In OT-CEC-UV profile of tryptic peptides of native BSA, 44 peaks could be resolved, whereas a reduced number of 38 peaks were observed in the profile of tryptic peptides of glucose-glycated BSA and only 30 peaks were found in the case of ribose-glycated BSA. The developed OT-CEC can be potentially used for monitoring of protein glycation.

Identification of collagen types in tissues using HPLC-MS/MS.

A method for the determination and quantification of collagen types I-V in rat tissues has been developed. This method is based on collagen fragmentation by cyanogen bromide followed by trypsin digestion. After that, HPLC-MS/MS (HPLC coupled to an IT mass spectrometer) analyses of the resulting peptide mixtures (peptide maps) were performed. Specific peptides for each collagen type were selected. According to online databases, these peptides are present in human, bovine, and rat collagens. As a result, this method can be potentially applied to other species' tissues as well, such as human tissues, and provides a universal and simple method of quantifying collagen types. The applicability of this method for analyzing collagen types was demonstrated on rat tissues (skin, tendon, and aorta).

Study of posttranslational non-enzymatic modifications of collagen using capillary electrophoresis/mass spectrometry and high performance liquid chromatography/mass spectrometry.

The depository effects that occur in slowly metabolized proteins (typically glycation) are very difficult to assess, owing to their extremely low concentration in the protein matrix. Collagen accumulates reactive metabolites through reactions that are not regulated by enzymes. A typical example of these non-enzymatic changes is glycation (the Maillard reaction, the formation of advanced glycation end products), resulting from the reaction of the oxo-group of sugars with the epsilon-amino group of lysine and arginine. Collagen samples (type I) as a test protein were incubated separately with glucose, ribose and malondialdehyde. Collagen was fragmented with cyanogen bromide and then digested with trypsin. This peptide digest was separated by CE, CE-MS/MS, and HPLC-MS/MS. An ion trap MS was used and MS conditions were optimized for both methods. These on-line CE-MS/MS and HPLC-MS/MS couplings made it possible to discover specific modifications such as (N(epsilon)-(carboxymethyl)-lysine) in the precise location in the structure of collagen corresponding to posttranslational non-enzymatic modifications. A new CE-MS/MS technique for peptide analysis was developed, and applied in the identification of posttranslational modifications in slowly metabolized test proteins.

Optical sensing system for ATP using porphyrin-alkaloid conjugates.

Tetrabrucin-porphyrin as a sensor for ATP was designed and tested; selectivity for ATP was proved in the presence of ADP and AMP.

Ring-substituted benzohydroxamic acids: 1H, 13C and 15N NMR spectra and NH-OH proton exchange.

NMR spectra (1H, 13C, 15N) of para- and meta-substituted benzohydroxamic acids were studied in dry dimethyl sulfoxide solutions. The 13C chemical shifts were very close to those found by cross-polarization magic angle spinning in solids, the hydroxamic (not hydroximic) structure of which is unambiguous. The hydroxamic structure of these acids in DMSO solutions was proved independently by their 15N chemical shifts. The 15N and 1H chemical shifts of the NH-OH fragment showed excellent mutual dependences and dependences on the nature of the ring substituent. According to these dependences and ab initio energy calculations, all the acids assume the same Z conformation. Proton exchange between hydroxamic OH and NH groups in DMSO proceeded by both intra- and intermolecular exchange and the rates did not exhibit any simple relationship to the substituent constants.