Determination of the post-translational modifications of salivary acidic proline-rich proteins.

Human salivary acidic proline-rich proteins were analyzed by electrospray-ion trap mass spectrometry and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All acidic-PRP isoforms share a common N-terminal region, which contains a pyroglutamic acid residue at the N-terminus, and two phosphorylation sites on Ser 8 and 22. At the same time, HPLC-MS spectra revealed isoforms of PRP-1 and PRP-3 having a different number of phosphoserine residues, namely, a mono-phosphorylated form of PRP-1 and PRP-3 and a tri-phosphorylated form of PRP-1. The analysis of the masses of tryptic digests suggested that the third phosphate residue should be located on Ser 17. Another protein with a mass of 30,923 amu was detected along the HPLC pattern and MS data of its tryptic digest suggested that it corresponds to the dimer of Pa, the isoform of PRP-1 with a substitution Arg-Cys at 103 position. Finally, structural identification is pending for another post-translational modification of acidic-PRP that provides an increase of 111-114 amu.

Determination of S-nitrosoglutathione in erythrocytes by capillary zone electrophoresis.

A method for separation and quantification of S-nitrosoglutathione in red cell extracts by capillary electrophoresis is reported. The method is based on the direct analysis of the metaphosphoric acid erythrocyte extract containing diethylenetriaminepentaacetic acid. Optimization of the method is briefly discussed. Best results in the shortest time were obtained at 25 degrees C, using a coated capillary, 7 kV applied voltage and phosphate sodium 40 mmol/L (pH 2.2) as running buffer. Reproducibility, detection limits, and recoveries of S-nitrosoglutathione analyses were checked. The results evidenced that S-nitrosoglutathione is formed in erythrocytes treated with S-nitrosocysteine, a transnitrosating agent. Under our experimental conditions, the contemporaneous detection and quantification of reduced and oxidized glutathione present in cell extract could also be performed.

Determination of biophysical parameters of polypeptide retro-inverso isomers and their analogues by capillary electrophoresis.

The relationship between the electrophoretic mobility, microobs, Stokes radius, rs, ionization state, and solution conformation of the all L-alpha-polypeptide, 1, the corresponding retro-all D-alpha-polypeptide, 2, and several truncated analogues, 3-5, has been investigated under low pH buffer conditions by high-performance capillary zonal electrophoresis (HPCZE) with coated capillaries. The results confirm that, under these conditions, the all L-alpha-polypeptide, 1, and its retro-inverso isomer, 2, exhibit nonidentical electrophoretic mobilities and thus different Stokes radii. At higher pH values, i.e., pH 5.0, the electrophoretic behavior of this retro-inverso isomer pair, however, converges. These results indicate that variations in the dipole characteristics of the polypeptide main chain and subtle differences introduced by the spatial constraints of the L-alpha-Pro-->D-alpha-Pro residue replacement lead to differences in the Stokes radii and electrophoretic mobilities of these polypeptides. Since the observed electrophoretic mobilities, microobs, reflect the mean of the mobilities of each charge species participating according to their Stokes radius or their intrinsic charge and mole fraction abundances, the results confirm that polypeptide retro-inverso isomers with unmodified amino and carboxy termini are resolvable. This outcome was achieved despite their notional topographical and conformational similarities as assessed from high-field proton nuclear magnetic resonance (1H NMR) spectroscopy and circular dichroism (CD) spectroscopy.

The pH dependence of predictive models relating electrophoretic mobility to peptide chemico-physical properties in capillary zone electrophoresis.

We applied best fitting procedures to capillary electrophoresis (CE) mobility values, measured at varying acidic pH, of a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da. This method allowed the contemporary measurements of C-terminus and carboxylic group of the side-chain of aspartic and glutamic acid dissociation constants and of peptide Stokes radius at different protonation stages. Stokes radius was related to peptide molecular mass M at the power of a fractional coefficient, and best correlation was found at pH 2.25, the fractional coefficient being equal to 0.68. This value is close to that proposed by R. E. Offord (Nature 1966, 211, 591-593), who suggested a proportionality between the polymer Stokes radius and M(2/3). The coefficient value decreases at higher pH, reaching a value of 0.58 at pH 4.25, corresponding to a mean peptide conformational transition towards more compact structures as a consequence of C-terminus dissociation. The measurement of the dissociation constants of each peptide allowed us to determine the percentage error on peptide charge predictions performed utilizing mean dissociation constants. Even for the charge, the best predictive performance is obtained at the most acidic edge of the range of the pH studied, mainly at pH 2.25. Conclusively, this study shows that the best performance of predictive models for peptide CE mobility is obtainable in the very acidic pH range (2.25-2.50) and in the absence of electroosmotic flow, and that a satisfactory predictive equation of peptide electrophoretic mobility (m2V(-1)s(-1) is given by mu = 85.4(Z/M(0.68))10(-8).

Predictive model for capillary electrophoretic peptide mobility in 2,2,2-trifluoroethanol-water solution.

Using capillary electrophoresis (CE) on a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da, the Stokes radii at different protonation stages and the acidic dissociation constants in water and in a 2,2,2-trifluoroethanol (TFE) water mixture (30% v/v) were determined. These results permitted us to establish separately the reliability of semiempirical models utilized for the prediction of peptide size and charge at different acidic pHapp (pHapp range: 2.00-4.25). The data obtained on size and charge were utilized in order to provide suitable mobility predictions on the basis of the charge-to-size ratio. The best predictive conditions for size and charge were found at the most acidic range of pHapp studied (2.00-2.25), either in water or a TFE-water mixture, and reliable predictive equations for peptide mobility were established at this pHapp.

Peptide analysis by capillary (zone) electrophoresis.

In this review various aspects concerning the application of capillary (zone) electrophoresis for peptide analysis will be critically examined. First, the basic instrumental requirements of CE apparatus and the strategies employed to enhance sensitivity in the analysis of underivatized sample are described. Multidimensional separative techniques of complex peptide mixtures that use CE as final step and the coupling of CE with mass spectrometry are subsequently discussed. A theoretical section describes the relationships existing between peptide mobility and the pH of the separation buffer. These relationships evidence that proton dissociation constants and Stokes radius at different protonation stages can be calculated by measuring the electrophoretic mobility at different pH values. Investigation of peptide mobility dependence on pH allows us to establish the optimum conditions, in terms of resolution, for peptide separation. Subsequently, a critical discussion about semiempirical models predicting peptide mobility as a function of chemico-physical peptide properties is presented. A section is devoted to the description of principles of peptide affinity capillary electrophoresis, underlining the similarity with peptide-proton interaction. CE separations performed in aquo-organic solvents are also critically discussed, showing the good performance obtained by using water-2,2,2-trifluoroethanol solutions. Finally, selected CE applications for the determination of peptide chemico-physical properties and conventional analysis, like peptide mapping, are reported.

Determination of peptide dissociation constants and Stokes radius at different protonation stages by capillary electrophoresis.

Peptide electrophoretic mobility measured by capillary zone electrophoresis can be regarded as deriving from the mean of mobilities of different protonated forms, each one participating according to its charge. Stokes radius and relative percentage. The percentage is a function of the peptide dissociation constant and solution pH. Therefore, mobility modifications due to pH variations can be related to peptide dissociation constant, charge, and Stokes radius throughout general binding equations. Thus, not only can peptide dissociation constants be measured, but information about Stokes radius modifications linked to proton loss can also be obtained with picomoles of peptide.

Effect of 2,2,2-trifluoroethanol on capillary zone electrophoretic peptide separations.

The use of 2,2,2-trifluoroethanol-water mixtures for peptide separations by capillary zone electrophoresis (CZE) displays some advantages over aqueous solutions. First, the increase in viscosity reduces and stabilizes the running current and facilitates heat dispersion, with a consequent improvement in the number of theoretical plates. Second, the decrease in the dielectric constant leads to a modification of the dissociation constants of the ionizable groups. The consequence is a change in selectivity that, for several favourable peptide pairs, provides an increase in resolution. Third, the interaction trifluoroethanol with the peptide modifies the Stokes radius in a manner strongly dependent on the peptide sequence. This can also be utilized for an increase in CZE performance. Fourth, the structural properties of 2,2,2-trifluoroethanol are particularly useful for an improvement in the separation of large apolar peptides. Finally, the use of trifluoroethanol strongly stabilizes the capillary coating.

The use of capillary electrophoresis for the determination of hemoglobin variants.

The application of capillary electrophoresis and related techniques for the detection of hemoglobin variants is described. Capillary zone electrophoresis (CZE) was applied for the analysis of intact tetrameric hemoglobin. CZE under denaturing conditions was used for the separation of globin chains. Both CZE and micellar electrokinetic capillary chromatography were applied for a fast and sensitive separation of tryptic digests and for the analysis of amino acid derivatives.

Capillary zone electrophoresis of peptides: prediction of the electrophoretic mobility and resolution.

The determination of the pKa values of some selected peptides of similar size was performed by microtitration, which makes possible an accurate determination of the peptide charge as a function of the solution pH. Capillary zone electrophoresis separation of these peptides on modified capillaries at acidic pH showed that the electrophoretic mobility correlates with the peptide charge. This observation suggests that when an appropriate charge value is used, the basic electrophoretic equation is respected and, at least at a peptide charge value less than 1, the utilization of alternative semi-empirical predictions is not necessary. As a general rule, a peptide separation at acidic pH values is to be preferred to that at basic pH values. In fact, at basic pH a separation in the absence of both electroosmotic flow and of spurious interactions between the peptides and the inner wall of the capillary is difficult, owing to the instability of capillary modification. Further, from the differences in the peptide charge, a prediction of the best resolution as a function of the pH could be obtained; in fact, the resolution, for peptides of similar size and in the absence of electroosmotic flow, is connected to a simple equation, where the principal term depends on the effective charge of the peptides, which is a function of the pH of the solution and the pKa values of the peptides. The predictions of resolution at acidic pH agreed well with the experimental results; the spatial resolution measured in the separation of met- and leu-enkephalin was virtually coincident with the predicted resolution; in the case of a mixture of four model tetrapeptides of sequence GGNA, GGQA, GGDA and GGEA some anomalous results with respect to the predicted resolutions were observed. Nevertheless, an acceptable prediction can also be made in this case.

Separation of reduced and oxidized glutathione by micellar electrokinetic capillary chromatography.

The separation of reduced and oxidized glutathione at an absolute sensitivity of about 100 pg by micellar electrokinetic capillary chromatography without derivatization is described. The time required for the separation is less than 10 min (the time between two following injections is about 15 min). The separation is characterized by high efficiency and good reliability. A partition mechanism is responsible for the high resolution observed. The method was utilized for the analysis of commercial preparations of glutathione and a good agreement with the expected results was obtained; the oxidation of the commercial glutathione in solution was easily analysed.

Peptide mapping through the coupling of capillary electrophoresis and high-performance liquid chromatography: map prediction of the tryptic digest of myoglobin.

The tryptic map of horse myoglobin was analysed through capillary electrophoresis using capillaries modified by a monolayer of acrylamide. The results were reproducible and the map was obtained in less than 30 min from ca. 8 pmol of tryptic digest. The peptide identification was performed using peptides previously identified by high-performance liquid chromatography. The peak areas measured using the two techniques are closely related, and the comparison of elution and migration times shows that the two techniques provide different maps. Furthermore, using the semiempirical relationship suggested by Grossman et al. [Anal. Biochem., 179 (1989) 28], which links the electrophoretic mobility to the charge of the peptide and its number of amino acids, a good agreement between predicted and experimental mobilities was observed.

High-performance liquid chromatography in protein sequence determinations.

The use of reversed-phase high-performance liquid chromatography (RP-HPLC) for the determination of protein sequences is reported. Topics considered include the peptide separation of endoprotease digestion mixtures, the application of HPLC peptide mapping as an efficient system to check the accuracy of an assumed protein sequence, obtained indirectly by DNA sequencing and the use of HPLC for amino acid analysis in the Edman sequence strategy. The use of RP-HPLC for an unconventional sequence strategy is demonstrated; HPLC exopeptidase mapping appears to be particularly useful as a future technique for small terminal sequence analysis. Finally, the coupling of HPLC with fast atom bombardment mass spectrometry is discussed.

Determination of the non-enzymatic glycation of hemoglobin by isoelectrofocusing of its globin chains.

Micro cation exchange chromatography determination of HbA1c does not provide a complete picture of Hb glycation, for it does not determine all the glycated forms of hemoglobin. For the determination of total glycation, we describe here a rod IEF method, which allows the simultaneous quantitation of glycation on alpha and beta globin chains. The method exhibits good sensitivity; it is not affected by artifacts deriving from temperature, hypertriglyceridemia, Hb variants or labile HbA1 (aldiminic Hb). The results obtained indicate that in a normal population approximately 18% of the beta chain and 8% of the alpha chain are glycated. These mean percentages increase in the diabetic to 28% and 12%, respectively. The beta chain is glycated on both valine and lysine residues, while the alpha chain is glycated only on the latter. HbA1 values from micro cation exchange chromatography are significantly related to both alpha and beta glycation. Thus, valinic or lysinic glycation have roughly the same clinical significance.

Use of killer toxins for computer-aided differentiation of Candida albicans strains.

Killer toxins were isolated from eight selected killer yeasts. Their activity on 100 Candida albicans isolates of human and animal origin was studied. A computer aided system for differentiating C. albicans strains was developed. By using this system, it was possible to differentiate 14 biotypes of C. albicans isolates based on their susceptibility to the killer toxins.