Pavla Sedláková

Proteins of insoluble matrix of avian (gallus gallus) eggshell.

The protein composition of the insoluble avian eggshell matrix was studied. The determination of these proteins insoluble in water (EDTA-insoluble) was carried out using enzymatic cleavage followed by a high-performance liquid chromatography–mass spectrometry method. The influence of various enzymes on the protein splitting also was studied. The distribution of proteins depends on the type of layer (localization within the eggshell): ovocalyxin-32 was found mainly in the outer layer (the cuticle); ovocleidin-116 and 17 and ovocalyxin-36 were found throughout the whole eggshell, whereas ovalbumin was only found in the inner layer, the mammillary. The pigment (protoporphyrin IX) was mainly found in the cuticle and is incorporated into the protein network.

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.

Open-tubular capillary electrochromatography with bare gold nanoparticles-based stationary phase applied to separation of trypsin digested native and glycated proteins.

In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins.

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<ribose<glyoxal<glutardialdehyde). Carboxymethylation of lysine (CML) was the main type of modification detected. The HPLC/MS method achieved higher coverage and a larger amount of CML was identified compared to CZE/MS.

Separation of tryptic peptides of native and glycated BSA using open-tubular CEC with salophene–lanthanide–Zn2+ 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.

Determination and Quantification of Collagen Types in Tissues Using HPLC-MS/MS

A method for the determination and quantification of collagen types (I - V) using sample pretreatment has been developed. This work is a continuation of our previous work dealing with the determination of collagen types I and III in tissues (1). The tissues (rat placenta and porcine cartilage) were firstly homogenized with a pestle in a grinding mortar with liquid ni- trogen. Collagens were isolated from these tissues by cleavage with pepsin. The collagen types of interest were then pre- cipitated successively by adding sodium chloride. For quantitation purposes, the sample preparation protocol has been simplified to the one-step precipitation of collagens from a solution containing 4.5 mol/L of sodium chloride. The frac- tions were fragmented by cyanogen bromide and digested with trypsin. After that, HPLC-MS/MS (high performance liq- uid chromatography coupled to an ion trap mass spectrometer) analyses of the resulting peptide mixtures (peptide maps) were performed. Based on these analyses, specific (marker) peptides for each of the collagen types were selected. The marker peptides were then synthesized and used to identify and quantify the above-mentioned collagen types in tissues us- ing HPLC-MS/MS, and for determining the limits of detection and quantification.

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 C18 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.

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.

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.