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.

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.

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.

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

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).

Capillary electrochromatography of proteins and peptides.

This review summarizes applications of CEC for the analysis of proteins and peptides. This "hybrid" technique is useful for the analysis of a broad spectrum of proteins and peptides and is a complementary approach to liquid chromatographic and capillary electrophoretic analysis. All modes of CEC are described--granular packed columns, monolithic stationary phases as well as open-tubular CEC. Attention is also paid to pressurized CEC and the chip-based platform.

Voltage-assisted capillary LC of peptides using monolithic capillary columns prepared by ring-opening metathesis polymerization.

We examined the use of monolithic capillary columns prepared via ring-opening metathesis polymerization (ROMP) for peptide separation in voltage-assisted capillary LC (voltage-assisted CLC). In order to demonstrate their potential for peptide separation, ROMP-derived monoliths with RP properties were prepared. The preparation procedure of monoliths was transferred from ROMP monoliths optimized for CLC. ROMP monoliths were synthesized within the confines of 200 microm id fused-silica capillaries with a length of 37 cm. After optimization of the chromatographic conditions, the separation performance was tested using a well-defined set of artificial peptides as well as two peptidic mixtures resulting from a tryptic digest of BSA as well as a collagenase digest of collagen. ROMP monoliths showed comparable performance to other monolithic separation media in voltage-assisted CLC published so far. Therefore, we conclude that by optimizing the composition of the ROMP monoliths as well as by using the controlled manner of their functionalization, ROMP monoliths bear a great potential in CLC and CEC.

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.

Matrices for capillary gel electrophoresis--a brief overview of uncommon gels.

This article gives an overview of uncommon replaceable matrices (gels) for capillary gel electrophoresis. This electrophoretic technique is useful mainly for the separation and analysis of biopolymers-nucleic acids and their fragments, and proteins/peptides. Commonly used gels are not reviewed. Those mentioned and discussed here are gels containing saccharides, newly developed acrylamide-based gels and thermoadjustable viscosity polymers, namely triblock copolymers and grafted polyacrylamide.

Capillary electromigration methods for the study of collagen.

This review paper gives an overview of capillary electromigration methods used in the analysis of collagen. Analyses of the parent chains as well as of the bromcyane and collagenase fragments of collagens are presented. Methods include capillary zone electrophoresis, capillary gel electrophoresis, micellar electrokinetic chromatography as well as combinations of HPLC and capillary electrophoresis, and capillary electrophoresis with mass spectrometry.