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.     

Proteomic identification of technologically modified proteins in malt by combination of protein fractionation using convective interaction media and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for the fast separation of proteins and identification of their modifications based on the use of monolithic chromatographic media and mass spectrometric techniques is described. This method has been developed and applied to the analysis of malt proteins and its posttranslational modifications (glycation). Glycation, one of the most common forms of posttranslational modifications (PTM), can be detected in both biological and industrial samples. Our attention was focused on the investigations of possible chemical modifications of water-soluble barley proteins during malting process by combination of anion-exchange chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The malt extract was directly fractioned by anion-exchange chromatography using short monolithic columns and a linear gradient from 0 to 700 mM NaCl. Sufficient fractionation was obtained for malt sample, which demonstrates the potential of anion-exchange chromatography on this type of column. Proteins in separated fractions were identified by MALDI-TOF/TOF MS. Our proteomic analysis provided the identification of the major proteins present in the malt that were found to be heterogeneously glycated after malting. One of these proteins: nonspecific lipid transfer protein 1 (LTP1) can be used as a marker for characterization of glycation during malting. This protein and its modifications can be easily determined by the developed method.     

Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV

Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement membranes. Human collagen type IV was modified via incubation with 200 mM NaNO(2) (pH=7.38) for one week at 37(o)C. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C(12) RP column. Site specificity was determined from tandem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical R(t), lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(IV)Y1081. These sites are located in the triple-helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV.     

Open tubular capillary electrochromatography: A useful microreactor for collagen I glycation and interaction studies with low-density lipoprotein particles

Diabetes, a multifunctional disease and a major cause of morbidity and mortality in the industrialized countries, strongly associates with the development and progression of atherosclerosis. One of the consequences of high level of glucose in the blood circulation is glycation of long-lived proteins, such as collagen I, the most abundant component of the extracellular matrix (ECM) in the arterial wall. Glycation is a long-lasting process that involves the reaction between a carbonyl group of the sugar and an amino group of the protein, usually a lysine residue. This reaction generates an Amadori product that may evolve in advanced glycation end products (AGEs). AGEs, as reactive molecules, can provoke cross-linking of collagen I fibrils. Since binding of low-density lipoproteins (LDLs) to the ECM of the inner layer of the arterial wall, the intima, has been implicated to be involved in the onset of the development of an atherosclerotic plaque, collagen modifications, which can affect the affinity of native and oxidized LDL for collagen I, can promote the entrapment of LDLs in the intima and accelerate the progression of atherosclerosis.In this study, open tubular capillary electrochromatography is proposed as a new microreactor to study in situ glycation of collagen I. The kinetics of glycation was first investigated in a fused silica collagen I-coated capillary. Dimethyl sulphoxide, injected as an electroosmotic flow marker, gave information about the charge of coating. Native and oxidized LDL, and selected peptide fragments from apolipoprotein B-100, the protein covering LDL particles, were injected as marker compounds to clarify the interactions between LDLs and the glycated collagen I coating. The method proposed is simple and inexpensive, since only small amounts of collagen and LDL are required. Atomic force microscopy images complemented our studies, highlighting the difference between unmodified and glycated collagen I surfaces.     

In vitro nonenzymatic glycation of DNA nucleobases: an evaluation of advanced glycation end products under alkaline pH

The advanced glycation end products (AGEs) of DNA nucleobases have received little attention, perhaps due to the fact that adenine, guanine, cytosine and thymine do not dissolve under mild pH conditions. To maintain nucleobases in solution, alkaline pH conditions are typically required. The objectives of this investigation were twofold: to study the susceptibility of DNA nucleobases to nonenzymatic attack by different sugars, and to evaluate the factors that influence the formation of nucleobase AGEs at pH 12, i.e., in an alkaline environment that promotes the aldo–keto isomerization and epimerization of sugars. Varying concentrations of adenine, guanine, thymine and cytosine were incubated over time with constant concentrations of D-glucose, D-galactose or D/L-glyceraldehyde under different conditions of temperature and ionic strength. Incubation of the nucleobases with the sugars resulted in a heterogeneous assembly of AGEs whose formation was monitored by UV/fluorescence spectroscopy. Capillary electrophoresis and HPLC were used to resolve the AGEs of the DNA adducts and provided a powerful tool for following the extent of glycation in each of the DNA nucleobases. Mass spectrometry studies of DNA adducts of guanine established that glycation at pH 12 proceeded through an Amadori intermediate.     

Voltammetric response of phenylboronic acid monolayer-modified gold electrode to sugars.

The surface of a gold (Au) disk electrode was modified with a self-assembled monolayer consisting of phenylboronic acid moiety to fabricate a voltammetric sensor sensitive to sugars. The modified Au electrode exhibited a voltammetric response to sugars in the presence of Fe(CN)6(3-) ion in the sample solution at neutral pH. The peak current of the cyclic voltammograms decreased depending on the type and concentration of sugars. The dynamic range of the electrode is 3 - 100 mM for glucose and mannose and 1 - 30 mM for fructose. The sugar sensor can be used repeatedly after rinsing in 10 mM acetate buffer (pH 4.5).     

Dependence of cyano bonded phase hydrolytic stability on ligand structure and solution pH

As part of our program to develop more stable cyano (CN) high-performance liquid chromatography (HPLC) column packings, we have evaluated hydrolytic stability as a function of ligand connectivity, chain length, and side group steric protection and the pH of the mobile phase. Three accelerated tests were used to evaluate stability: (1) A non-HPLC screening test measuring carbon loss in refluxing MeOH-100 mM KH2PO4 pH 4.5 (1:1, v/v) solution; (2) a continuous flow HPLC test measuring capacity factor maintenance in 1% trifluoroacetic acid in water (pH 1.02) at 80 degrees C; and (3) a continuous flow HPLC test measuring column efficiency maintenance in 50 mM triethylamine in water (pH 10.00) at 50 degrees C. The stability of the CN phases was found to be dependent on both ligand chemical structure and the pH of the test conditions. The starting screen test of intermediate pH was least able to differentiate the CN phases based on structure, because two different degradation mechanisms appear to offset each other (acid induced siloxane bond cleavage vs. base induced silica dissolution). A trifunctional and a sterically protected CN phase were notably stable under the acidic test conditions, but had poor stability under basic conditions. Conversely, chain extension afforded poor stability under acidic conditions, but did afford improved stability at higher pH. In total, the data indicate that good CN column stability can be achieved by using a trifunctional or a sterically protected phase in acidic mobile phases. However, as mobile phases of intermediate or higher pH are employed, shorter column lifetimes can be expected due to an accelerated dissolution of the underlying silica substrate. Materials were also compared chromatographically using a mixture of non-polar, polar, and basic analytes under reversed-phase conditions.     

Mineralization of biomimetically carboxymethylated collagen fibrils in a model dual membrane diffusion system

In the present work, we show for the first time, that N^?-carboxymethyllysine is the major product of the in vitro non-enzymatic glycation reaction between fibrillar collagen and glucuronic acid. Dual diffusion membrane system was effectively used for oriented crystal growth of octacalcium phosphate/hydroxyapatite on the biomimetically carboxymethylated collagen fibrils. We hypothesize that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions in such a way that a critical nucleus of ions can be formed, leading to the formation of the mineral under specific micro-environment conditions achieved by using diffusion membrane system.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry

Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH]+), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H]4+) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H]4+, which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation.     

Evaluation of peptide electropherograms by multivariate mathematical-statistical methods. I. Principal component analysis.

Depository effects in slowly metabolised proteins, typically glycation or the estimation of products arising from the reaction of unsaturated long-chain-fatty acid metabolites (possessing aldehydic groups) are very difficult to assess owing to their extremely low concentration in the protein matrix. In order to reveal such alterations we applied deep enzymatic fragmentation resulting in a set of small peptides, which, if modified, are likely to change their electrophoretic properties and can be visualised on the resulting profile. Peptide maps of collagen (a mixture of collagen types I and III digested by bacterial collagenase) were applied as the model protein structure for detecting the nonenzymatic posttranslational changes originating during various physiological conditions like high fructose diet and hypertriglyceridemic state. Capillary electrophoresis in acidic media (sodium phosphate buffer, pH 2.5) was used as the separation method capable of (partial) separation of over 60 peptide peaks. Two to 13 changes were revealed in the profiles obtained reflecting the physiological conditions of the animals tested. Combination of peptide profiling with subsequent t-test evaluation of individual peak areas and principal component analysis based on cumulative peak areas of individual sections of the electropherograms allowed to determine in which section (part) of the electropherogram the physiological state indicating changes occurred. Simultaneously it was possible to reveal the qualitative differences between the four physiological regimes investigated (i.e., which regime affects the collagen molecules most and which affects them least). The approach can be used as guidance for targeted preseparation of the very complex peptide mixture.     

Silver staining of collagen type I after sodium dodecylsulphate polyacrylamide gel electrophoresis: effect of Maillard reaction.

Differences in the acidic silver staining, after sodium dodecylsulphate polyacrylamide gel electrophoresis, between the alpha 1 and alpha 2 collagen chains, as well as between rat-tail tendon and calf-skin collagen type I, were observed. The staining conditions at which the staining differences are both most expressed and reproducible were characterized. Age differences between staining of the alpha 1 CB6 fragment from young rats (2 and 12 months) and old rats (29 months) indicated that different susceptibilities of collagen species to the silver staining can be the result of different extents of some age-dependent post-translational modification, such as glycation. In vitro incubation of acid-soluble rat-tail tendon collagen with various sugars led to an increase in silver staining compared with samples incubated in the absence of sugar. This effect was inhibited by sodium cyanoborohydride, diethylenetriamine pentaacetic acid and aminoguanidine, i.e. compounds inhibiting the Maillard reaction at various stages. It can be concluded that the enhanced silver susceptibility of glycated collagen is related to advanced-phase Maillard reaction products attached to collagen.     

Analysis of formose sugar and formaldehyde by high-performance liquid chromatography

Formose sugar and formaldehyde (HCHO) were analyzed using high-performance liquid chromatography (HPLC) utilizing a CarboPac PA1 column (Dionex) and pulsed amperometric detection. This HPLC system was unsuitable for the analysis of formose sugar and HCHO and thus reducing sugars and unconverted HCHO were determined by endowing them with charges through a derivatization method using 2,4-dinitrophenylhydrazine. The separation and detection of compounds were performed by three Chromolith RP-C18 columns (Merck) and diode array detection, at a wavelength of 360 nm ultraviolet light, respectively. Lower sugars (except HCHO) showed some instabilities when the derivatized samples were kept for the extended periods of time. For C₅ and consecutive higher sugars, a certain derivatization time was necessary. In the present case (formose reaction with partial HCHO conversion), approximately 18 h may be a reasonable compromise for the derivatization reaction. A derivatization agent to compound mole ratio of up to 100:1 was required to complete the derivatization of C₄ and higher sugars. However, the analysis of C₄ and consecutive higher sugars is problematic for example due to overlapping of peaks or branched-chain sugars.     

Analysis of oligomeric and monomeric carbohydrates from hydrothermal degradation of cotton-waste materials using HPLC and GPC

Summary The hydrolysis of different cotton materials under hydrothermal conditions was investigated. For the analysis of the resulting reaction products high-performance liquid chromatography and gel-permeation chromatography were applied. HPLC columns with ion exchange materials as stationary phases (HPX 87P and Shodex S801) enabled the separation of monomeric sugars and degradation products. Oligomeric sugars were analyzed using a gel column (Bio-Gel P-2) as well as a HPLC column (HPX 42A). Through identification of additional reaction products the mass balance of the hydrolysis can be improved and the analysis of the distribution of the oligomeric sugars gives information for the selection of the reaction conditions.     

Trace analysis of sugars by HPLC and post-column derivatization

Summary A HPLC system with post-column derivatization for the quantitative analysis of sugars in complex matrices is described. As reagent a 0.2% solution of thymol in concentrated sulfuric acid has been used. The reaction is sensitive with reducing as well as non reducing sugars whereas sugar alcohols are discriminated. With this reagent and separation of sugars at high pH values with an anion exchange column it is possible to detect sugars in the ng range. Hence, it is possible to characterize wines not only by their fructose and glucose content but also by differences in the distribution of the other not fermentable sugars like trehalose, arabinose, galacturonic and glucuronic acid.     

Analysis of tofisopam in human serum by column-switching semi-micro high-performance liquid chromatography and evaluation of tofisopam bioequivalency

A rapid and sensitive column-switching semi-micro HPLC method is described for the direct analysis of tofisopam in human serum. The sample (100 microL) was directly injected onto the precolumn (Capcell Pak MF Ph-1), where unretained proteins were eluted to waste. Tofisopam was then eluted into an enrichment column using 13% acetonitrile in 50 mM phosphate buffer (pH 7.0) containing 5 mM sodium octanesulfonate and subsequently into the analytical column using 43% acetonitrile in 0.1% phosphoric acid containing 5 mM sodium octanesulfonate. The detection limit (2 ng/mL), good precision (CV < or = 4.2%) and speed (total analysis time 24 min) of the present method were sufficient for drug monitoring. This method was successfully applied to a bioequivalence test of two commercial tofisopam tablets.     

Influence of column length and pore size on high-performance ion-exchange chromatography of estrogen and progestin receptors

A high-performance liquid chromatographic (HPLC) procedure has been evaluated to establish a routine test in the clinical laboratory for measuring the profiles of estrogen and progestin receptor isoforms in human breast and endometrial tumors. This procedure will be used to determine if there is a relationship between particular isoform profiles and response to various endocrine therapies. Evaluation of various HPLC modes has shown that high-performance ion-exchange chromatography (HPIEC) with silica-based anion exchangers offers a promising approach. In this paper, we have compared HPIEC columns of different lengths (10 and 25 cm) and pore sizes (300, 500 and 1,000 A) in order to obtain an optimal separation procedure. Because of receptor lability, all investigations were performed at 4 degrees C. The mobile phase consisted of 10-500 mM phosphate buffer, supplemented with the stabilizing agent, sodium molybdate at pH 7.4. Recoveries from each of the columns were between 70-100%. The length of the column did not influence significantly the retention time and salt concentration required for elution of receptor proteins. However, pore sizes appeared to alter these parameters. With a larger pore size (1,000 A), the retention of proteins was lower (elution with 50 mM phosphate) than that observed with the 500-A pore size column (elution with 100 mM phosphate) or of the 300-A pore size column (elution with 150 mM phosphate). Based solely on recovery patterns and peak shape, we conclude that separation of receptor isoforms on a 1,000-A, 25-cm column is best suited for clinical analysis.     

Measurement of sugars and starches in foods by a modification of the AOAC total dietary fiber method.

A separation scheme for the determination of sugars and starch in processed food was developed. It is based on AOAC Method 985.29 for total dietary fiber with these modifications: carbohydrate starches are separated into soluble and insoluble fractions before they are hydrolyzed; acetonitrile is used instead of ethanol to separate sugars from enzyme-resistant carbohydrates, proteins, and other macromolecules; and a solid-phase extraction filter is included to remove substances that interfere with high-performance liquid chromatography (HPLC). Recovery studies indicate a > 97% sugar recovery. Twenty foods were analyzed. After enzymatic hydrolysis, fructose, glucose, sucrose, maltose, and lactose were extracted and determined by HPLC using a refractive index detector. Starch content was calculated from the increase in the amount of glucose. The results were compared with values listed on the "Nutrition Facts" panel for that food. The analyzed amounts of sugars and starches were 73-96% of declared values.     

Pyridoxamine,a scavenger agent of carbohydrates

Pyridoxamine has been found to inhibit protein glycation and to avoid the formation of advanced glycation end‐products (AGEs). One of the mechanisms by which pyridoxamine can inhibit glycation involves the scavenger of carbonyl groups with glycation capacity. In this work, we conducted a kinetic study of the reactions of pyridoxamine with various carbohydrates under physiological pH and temperature. The reactions involving hexoses were found to give a tricyclic compound ( 5 ) in addition to pyridoxal and pyridoxine. Such a tricyclic compound inhibits the Amadori rearrangement and the formation of other carbonyl compounds with glycating properties. The reactions involving pentoses gave compound 7 and pyridoxal—by transamination of the Schiff base. The transamination reaction enhances the inhibitory action of pyridoxamine. The formation rate constants for the Schiff base, k₃, were found to be similar to those for the reactions of D ‐glucose with amino acids, which suggests competition between pyridoxamine and terminal amino residues in proteins for glycating sites in sugars. These constants are dependent on the electrophilic character of the carbonyl carbon in the carbohydrate. © 2007 Wiley Periodicals, Inc. 39: 154–167, 2007     

Determination of homotaurine as impurity in calcium acamprosate by capillary zone electrophoresis.

A method is reported which allows the quantification of homotaurine as an impurity in the drug. After addition of taurine as an internal standard, the sample is derivatised with fluorescamine at ambient temperature in 10 mM borate buffer, pH 9.2. The analytes are separated by capillary zone electrophoresis in a 31.2 cm (21 cm to the detector) x 100 microns I.D. fused-silica capillary at a potential of +7 kV and 25 degrees C. A 40 mM borate buffer, pH 9.2, is used as the electrolyte and detection is carried out at 205 nm. The validation tests showed that the method is reliable between 0.01% and 0.15% (m/m) of homotaurine with respect to the active drug. The limits of quantitation (0.01%, m/m) and detection (0.004%, m/m) allows to control the homotaurine content of the drug substance for which the maximum tolerated level is 0.05% (m/m). The proposed procedure (derivatisation and separation) developed in CE is rapid (20-25 min) by comparison to that currently used in HPLC (75 min). Satisfactory agreement was found between several batches of acamprosate analysed by CE and HPLC.