Identification of the nitration site of insulin by peroxynitrite.

Our previous investigation indicated that insulin can be nitrated by peroxynitrite in vitro. In this study, the preferential nitration site of the four tyrosine residues in insulin molecule was confirmed. Mononitrated and dinitrated insulins were purified by RP-HPLC. Following reduction of insulin disulfide bridges, Native-PAGE indicated that A-chain was preferentially nitrated. Combination of enzymatic digestion of mononitrated insulin with endoproteinase Glu-C, mass spectrometry confirmed that Tyr-A14 was the preferential nitration site when insulin was treated with peroxynitrite. Tyr-A19, maybe, was the next preferential nitration site. According to the crystal structure, Tyr-B26 between the two tyrosine residues in insulin B-chain was likely easier to be nitrated by peroxynitrite.     

Detection of glycation sites in proteins by high-resolution mass spectrometry combined with isotopic labeling

The products of nonenzymatic glycation of proteins are formed in a chemical reaction between reducing sugars and the free amino group located either at the N terminus of the polypeptide chain or in the lysine side chain. Glycated proteins and their fragments could be used as markers of the aging process as well as diabetes mellitus and Alzheimer’s disease, making them an object of interest in clinical chemistry. In this article, we propose a new method for the identification of peptide-derived Amadori products in the mixtures obtained by enzymatic hydrolysis of glycated proteins. Two proteins, ubiquitin and human serum albumin (HSA), were modified with an equimolar mixture of glucose and [¹³C₆]glucose and were subjected to enzymatic hydrolysis. The obtained enzymatic digests were analyzed by high-resolution mass spectrometry (HRMS), and the peptide-derived Amadori products were identified on the basis of specific isotopic patterns resulting from ¹³C substitution. The number of glycated peptides in the digest of HSA detected by our procedure was in agreement with the data recently reported in the literature.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

The lysine glycation 1. A preliminary investigation on the products arising from the reaction of protected lysine and D-glucose

Summary The nature of the products arising from a 10 days, sterile incubation at 37°C and pH 7.2 of a 1:1 mixture of N--(p-tosyl)-lysine-methylesterhydrochloride and anhydrous D-glucose was investigated by fast atom bombardment mass spectrometry and¹H and¹³C nuclear magnetic resonance spectroscopies. Differently to the reactivity usually described on the basis of other analytical techniques, FAB mass spectrometric measurements indicate the occurrence of the reaction of protected lysine with more than one D-glucose molecule.     

Characterization of glycation in an IgG1 by capillary electrophoresis sodium dodecyl sulfate and mass spectrometry

We report a case study of characterization of a non-enzymatically glycated IgG1 using reducing capillary electrophoresis sodium dodecyl sulfate (CE–SDS) and mass spectrometry (MS). Glycation was found to occur nonspecifically at multiple sites in both the light and heavy chains. The glycated light and heavy chains result in wider peaks eluting late in the reducing CE–SDS profile; in particular, the glycated light chain behaved as a shoulder peak detected by either ultraviolet (UV) or laser-induced fluorescence (LIF) signals. The glycated species can be enriched by boronate affinity chromatography. Analyzing the enriched samples by reversed phase high-performance liquid chromatography in line with time-of-flight MS (RP–HPLC–TOF/MS) revealed adducts of +162 and +324 Da to both the light and heavy chains, suggesting the presence of multiple glycation sites. Tryptic peptide mapping and tandem mass sequencing were used to identify two glycation sites on each of the light and heavy chains.     

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.     

The effect of non-enzymatic glycation on the unfolding of human serum albumin

We monitored the unfolding of human serum albumin (HSA) and glycated human serum albumin (gHSA) subjected to guanidine hydrochloride (GndHCl) by using fluorescence and circular dichroism (CD) spectroscopy. A two-state model with sloping baselines best described the Trp-214 fluorescence unfolding measurements, while a three-state model best described the far-UV CD unfolding data. Glycation of HSA increased the [D](50%) point by approximately 0.20M. This corresponded to an increase in the free energy of unfolding of gHSA relative to HSA of 2.6kJ/mol. The intrinsic fluorescence of Trp-214 in gHSA is 0.72 of that of HSA and the far-UV CD spectrum of gHSA is nearly identical to that of HSA. These results showed that glycation altered the local structure around Trp-214 while not significantly impacting the secondary structure, and this alteration translated into an overall change in the stability of gHSA compared to HSA.     

A study on in vitro glycation processes by matrix-assisted laser desorption ionization mass spectrometry

The number of glucose molecules condensed on glycated bovine serum albumin have been easily determined by means of matrix-assisted laser desorption/ionization mass spectrometry. Measurements were carried out on samples from incubation of the proteins with glucose at different concentrations (0.02 M, 0.2 M, 2 M and 5 M). A clear increase in molecular mass of BSA with respect to incubation time is detected. In contrast to what is observed with fluorescence, the plots of molecular mass increase vs. incubation time show tha occurrence of a steady state, corresponding to the complete saturation of all the protein sites against glucose. Comparison of fluorescence and molecular mass data reveals that some further reactions, different from condensation, must take place, which could be in principle either intramolecular or originated by reactivity of modified condensed gluocse moieties vs. free glucose.     

Insights into the degradation of human elastin by matrilysin-1

Human matrilysin-1 (MMP-7) is one of the most potent elastases besides macrophage elastase in the family of matrix metalloproteinases (MMPs). It has been reported to provide macrophages with the highest elastinolytic capacity and plays key roles in diseases such as emphysema and cancer. Describing the enzymatic turnover of matrix components helps to understand the molecular basis of disease processes. Hence, in this work, the cleavage behavior of MMP-7 with respect to its natural substrate human elastin was investigated using mass spectrometric (MS) techniques and molecular modeling. Elastin peptides in the range of 500-8000 Da released through the action of MMP-7 were analyzed and domains susceptible to proteolytic attack by MMP-7 were identified. MMP-7 was found to mainly cleave in N- and C-terminal regions of elastin’s precursor, which involves linkages in domains encoded by exons 2, 3, 5-7, 26, and 30-33. In contrast, only few cleavages were found in the central part of the precursor and no cleavages in regions in elastin that are involved in cross-linking. MMP-7 shows a strong preference for Leu in P₁′ and also accepts Val, Gly, and Pro at this position, whereas Ala is not preferred at P₁′. Analysis by molecular modeling revealed that not only the size of the amino acid residue in P₁′ but also the orientation of the neighboring P₁ residue and, thus, the orientation of the peptide bond that is cleaved influences the cleavage preference of MMP-7. Overall, this study provides an important insight into the degradation of human elastin by MMP-7 and may aid in the development of approaches to treat elastin-degrading diseases.     

Detergency effects of nanofibrillar amyloid formation on glycation of human serum albumin

The prolonged glycation of human serum albumin (HSA) results in significant changes in its structure. The identity of these structural changes and the influence of carbohydrates on these changes require further study. Here, we evaluated structural changes and amyloid formation of HSA upon incubation with Glc, Fru, or Rib. Fluorescence spectrophotometry, surface tension analysis, and transmission electron microscopy (TEM) were utilized to evaluate the structures of glycated HSA. The physicochemical properties including excess free energy, protein adsorption at the air-water interface, critical aggregation concentration (CAC), and surface activity indicated an increase in hydrophobicity and partial unfolding of HSA structure upon glycation. Thus, it appears that AGE products can act as detergents. Incubation of HSA with these sugars after 20 wks induced significant amyloid nanofibril formation. Together these results indicate that prolonged glycation of HSA is associated with a transition from helical structure to beta-sheet (amyloid formation).     

The isotopic exchange of oxygen as a tool for detection of the glycation sites in proteins

A nonenzymatic reaction of reducing sugars with the free amino group located at the N terminus of the polypeptide chain or in the lysine side chain results in glycation of proteins. The fragments of glycated proteins obtained by enzymatic hydrolysis could be considered as the biomarkers of both the aging process and diabetes mellitus. Here we propose a new method for the identification of peptide-derived Amadori products in the enzymatic digest of glycated proteins. The products of enzymatic hydrolysis of the model protein ubiquitin were incubated with H₂¹⁸O under microwave activation. We observed that at these conditions the Amadori compounds selectively exchange one oxygen atom in the hexose moiety. The characteristic isotopic pattern of Amadori products treated with H₂¹⁸O allows fast and convenient identification of this group of compounds, whereas nonglycated peptides are not susceptible to isotopic exchange.     

Characterization of Cholecystokinin from the Human Brain

Human forms of cholecystokinin have not previously been characterized chemically. In this study, we have extracted and purified the predominant molecular form of cholecystokinin present in human cerebral cortex. The peptide was characterized by amino acid analysis, automated peptide sequencing, and fast atom bombardment mass spectrometry. It appears to be identical to porcine cholecystokinin-octapeptide, with the sequence of Asp-Tyr(SO3)-Met-Gly-Trp-Met-Asp-Phe(NH2). This structural identity is consistent with the observations that the peptide in human brain and porcine cholecystokinin-octapeptide are recognized similarly by a battery of antisera to porcine cholecystokinin; that they coelute from several chromatographic systems, including gel filtration, ion exchange, and reversed-phase; and that they possess similar biological activities.     

Proteomic analysis of Tityus discrepans scorpion venom and amino acid sequence of novel toxins

The Venezuelan scorpion Tityus discrepans is known to cause human fatalities. We describe the first complete proteomic analysis of its venom. By HPLC 58 different fractions were obtained and 205 different components were identified by MS analysis. Components having molecular masses from 272 to 57 908 amu were found. Forty homogeneous components had their N-terminal amino acid sequence determined by Edman degradation, from which two new peptides named TdK2 and TdK3 (meaning T. discrepans (Td) K(+) channel toxins 2 and 3) were fully characterized. The first contains 34 amino acid residues with a molecular mass of 3451 amu, and the second has 36 amino acids with 3832 amu. Both peptides are tightly bound by three disulfide bridges. TdK2 was shown to block reversibly the Shaker B K(+)-channel expressed heterologously in Sf9 cells. The systematic number assigned to TdK2 is alpha-KTx-18.2 and that of TdK3 is alpha-KTx-18.3. Comparative analysis of the amino acid sequences found suggests that this venom contains peptides highly similar to those that block K(+) channels, as well as those that modify the gating mechanisms of Na(+) channels, found in other scorpions. Additionally, peptides similar to defensins were also identified.     

Validation of a method for monitoring the manufacture of human insulin

A method for monitoring the manufacture of human insulin by HPLC was developed. The method was validated by the estimation of its linearity, accuracy, precision, specificity, and robustness; the limits of detection and quantitative assessment were also determined. It was proven that HPLC analysis enables reliable and reproducible results to be obtained and can be used for monitoring insulin manufacture.     

Pyroglutamyl apelin-13 identified as the major apelin isoform in human plasma

Apelin is emerging as an important hormone regulator of cardiovascular homoeostasis and an important biomarker for heart failure. Apelin concentrations have historically been measured by immunoassays; however, reported apelin concentrations measured in healthy volunteers show a large disparity from a few picograms per milliliter (pg/ml) to several nanograms per milliliter (ng/ml). Apelin exists in several isoforms ranging in size from 12 to 36 residues, and immunoassays generally cannot distinguish the specific forms present. In this study, an optimized method for enriching apelin peptides with cation-exchange beads followed with mass spectrometry analysis is presented. Apelin peptides are labile in plasma at physiological conditions; however, by lowering the plasma pH to 4.5, the recovery of apelin peptides can be increased significantly. Through optimizing the cation-exchange extraction process, we improved the lower limit of detection for most of the apelin peptides monitored to a few pg/ml. Using the improved method, we detected pyroglutamyl apelin-13 [(pyr)apelin-13] as the major apelin isoform present in plasma from several healthy volunteers at concentrations ranging from 7.7 to 23.3 pg/ml.     

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.     

Identification and relative quantification of membrane proteins by surface biotinylation and two-dimensional peptide mapping

Membrane proteins play a central role in biological processes, but their separation and quantification using two-dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin-coated resin and proteolytically digested. The resulting peptides are analyzed by high-pressure liquid chromatography and mass spectrometry, thus yielding a two-dimensional peptide map. Matrix assisted laser desorption/ionization-time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA-spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial-cadherin/actin/catenin complex, revealing an increased accumulation of beta-catenin at 2% O(2) concentration.     

L-Arginine inhibits in vitro nonenzymatic glycation and advanced glycosylated end product formation of human serum albumin

Summary L-Arginine (Arg) has a structure similar to that of aminoguanidine (AG) and may inhibit glycation and advanced glycosylated end product (AGE) formation. Human serum albumin (HSA) (100mg/ml) was incubated for 2 weeks with glucose (200mM) at 37°C or with glucose and equimolar concentrations of Arg, N--acetyl Arg, or AG with or without 25mM diethylenetriaminepentaacetic acid (DTPA). In the absence of DTPA, electrospray ionization mass spectrometry showed a 70% reduction of covalently bound glucose in the presence of Arg and a 30% reduction with AG. Digestibility by trypsin of HSA incubated with glucose and Arg was similar to that of HSA incubated alone. This suggests less covalent modification of HSA in the presence of Arg as compared with the absence of Arg. When incubations contained DTPA, autoradiography showed less¹⁴C labeling of HSA subunits in the presence of Arg and AG. When the-amino group of Arg was blocked with an acetyl group, labeling was similar to that of HSA incubated with glucose, suggesting involvement of the-amino group in the inhibition. Fluorescence of HSA at ex₃₇₀ and em₄₄₀ was reduced with Arg, but AG was more effective than Arg. These results suggest that Arg, like AG, can inhibit glycation and AGE formation.Presented in part at the FASEB meeting, Atlanta, GA, 1991.     

The origin and control of ex vivo oxidative peptide modifications prior to mass spectrometry analysis

The origin and control of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine-, and tryptophan-containing peptides obtained from typical "in-solution" or "in-gel" proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels were found to be predominantly associated with the extensive ex vivo sample handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have been evaluated using a series of model synthetic peptides and standard tryptic protein digests. Essentially complete oxidation of methionine- and S-alkyl cysteine-containing peptides was achieved by reaction with 30% hydrogen peroxide/5% acetic acid at room temperature for 30 min. Under these conditions, cysteine was also converted to cysteic acid, while only limited oxidation of tryptophan to oxindolylalanine, and methionine and S-alkyl cysteine sulfoxides to their respective sulfones, were observed. Efficient reduction of methionine- and S-alkyl cysteine sulfoxide-containing peptides was achieved by reaction in 1 M dimethylsulfide/10 M hydrochloric acid at room temperature for 10 and 45 min, respectively. None of the reduction conditions evaluated were found to result in the reduction of oxindolylalanine, cysteic acid, or methionine sulfone.     

The in vitro glycation of human serum albumin in the presence of Zn(II)

Amino groups of human serum albumin (HSA) can react non-enzymatically with carbonyl groups of reducing sugars to form advanced glycation end products (AGEs). These AGEs contribute to many of the chronic complications of diabetes including atherosclerosis, cataract formation and renal failure. The current study focused on in vitro non-enzymatic reactivity of glyceraldehyde (GA) and methylglyoxal (MG) with HSA and evaluated the rate and extent of AGE formation in the presence of varied concentrations of Zn(II). At normal physiological conditions, GA and MG readily react with HSA. The presence of Zn(II) in HSA-GA or HSA-MG incubation mixtures reduced AGE formation. This finding was confirmed by UV and fluorescence spectrometry, HPLC techniques, and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF). HPLC studies revealed decreased adduct formation of the glycated protein in the presence of Zn(II). The inhibition of AGE formation was intense at elevated Zn(II) concentrations. The results of this study suggest that Zn(II) may prove to be a potent agent in reducing AGE formation.