Identification of protein phosphorylation sites within Ser/Thr-rich cluster domains using site-directed mutagenesis and hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry

We describe a method for the analysis of multi-site phosphorylation in serine/threonine (Ser/Thr)-rich protein sequences. Site-specific mutagenesis was used to introduce tryptic cleavage sites in the serine glutamine/threonine glutamine cluster domain (SCD) of the human checkpoint protein kinase (Chk2). The mutant proteins were shown to autophosphorylate on residues that are inducibly phosphorylated when mammalian cells are exposed to ionizing radiation (serine 33/35, serine 516, threonine 68 and threonine 432). Five Ser/Thr clusters within the SCD were flanked by arginine or lysine residues to produce tryptic peptides for nanospray liquid chromatography (nanoLC)/linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry. Phosphorylation sites were assigned using accurate-mass-driven analysis and interpretation of low-energy collision-induced dissociation spectra acquired in the ion trap. In addition to verifying known phosphorylation sites, seventeen novel sites were identified within the SCD of Chk2. The approach should be applicable to other O-linked post-translational modifications that occur in proteins with Ser/Thr-rich sequences.     

Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.     

Separation and analysis of peptides and proteins from Vipera lebetina snake venom

Our studies of Levantine viper venom have demonstrated that the venom is a rich source of biomedically important proteins and peptides. The venom contains metalloproteases: thrombolytic, fibrin-degrading lebetase, an endothelial cell apoptosis inducing metalloprotease (VLAIP), factor X activator (VLFXA); serine proteases: factor V activator, bradykinin-releasing serine proteases, β-fibrinogenase, α-fibrinogenase and chymotrypsin-like protease and different other enzymes such as phosphodiesterase, 5`-nucleotidase, ribonuclease, phospholipase A2s and L-amino acid oxidase. Among nonenzymatic components venom contains: nerve growth factor, vascular endothelial growth factor, disintegrins, C-type lectins.Here we report the isolation and characterization of proteins and peptides from Vipera lebetina venom using size exclusion, ion exchange, hydrophobic interaction and affinity chromatography, HPLC, UPLC and MALDI-TOF MS methods. N-terminal sequences and internal sequences of tryptic peptides of different proteins were determined using Edman sequencing and LC-ESI-MS/MS techniques. On the basis of fragmental sequences of proteins the oligonucleotides were designed and used as primers for cDNA cloning. Using cDNA library of the venom gland of a single snake the cDNAs coding proteins were cloned and sequenced. Protein sequences were deduced from cDNA sequences.The substrate specificity of venom proteases against insulin B-chain, bradykinin, substance P, and 6-10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α₂-macroglobulin bait region and pregnancy zone protein were studied using MALDI-TOF mass spectrometry technique.     

Thermal properties of some small peptides (N-acetyl-amino acid-N′-methylamides) with non-polar side groups

Temperatures and molar enthalpies of fusion of a series of uncharged small peptides, namely the methylamides of N-acetyl substituted glycine, α-amino-butyric acid, alanine, valine, norvaline, leucine, isoleucine, norleucine, and proline, were measured by differential scanning calorimetry (d.s.c.), and molar entropies of fusion were derived. Both l- and dl-compunds were taken into account for the chiral molecules. No solid-to-solid transitions were detected from room temperature to fusion except for N-acetyl-N′-methyl alaninamide. Comparisons were made with the values for the N-acetyl amides of the corresponding amino acids previously reported. Both l enantiomers and dl racemates of α-aminobutyric acid, alanine, valine and isoleucine methylamides displayed temperatures of fusion sharply increasing as a function of molar mass, whereas much lower values, in countertendency with their molar mass increase, were found for proline and leucine methylamides. The racemic dl crystals showed temperatures of fusion of about 40 °C lower than those of the corresponding pure l enantiomers, except for proline and leucine derivatives. The enthalpies and entropies of fusion also varied as a function of molar mass following a similar trend with that of temperatures of fusion, except for alanine derivatives which showed lower values than expected. The values of ideal solubility of solids at T = 298.15 K were estimated based on their temperatures and molar entropies of fusion. Results were discussed with reference to the packing patterns based on hydrogen bonding and hydrophobic interactions.     

A convenient procedure for the preparation of Garner’s aldehyde

The palladium/silane reduction of a serine thioester derivative allows for the preparation of enantiomerically pure Garner’s aldehyde from serine, in gram quantities within 24 h.     

The binding mode of picrotoxinin in GABAA-ρ receptors: Insight into the subunit’s selectivity in the transmembrane domain

The channel blocker picrotoxinin has been studied with GABA_A-ρ1 and GABA_A-ρ2 homology models based on the GluCl crystal structure. Picrotoxinin is tenfold more potent for GABA_A-ρ2 than for GABA_A-ρ1 homomeric channels. This intra-subunit selectivity arises from the unconserved residues at the 2’ sites, which are the essential molecular basis for both the binding and potency of picrotoxinin. The serine residues at the 2’ positions of the ρ2 channel are predicted to form multiple hydrogen bonds and hydrophobic interactions with picrotoxinin, whereas the proline residues in the 2’ positions of ρ1 channels are predicted to form only hydrophobic contacts with picrotoxinin. However, although the studied ρ1 P2’G, A, and V models form no hydrogen bonds with picrotoxinin, they may participate in several hydrophobic interactions, and the ligand may have distinctive binding modes with GABA_A-ρ mutant channels. Picrotoxinin has a lower Emodel value with ρ2 than ρ1 homomeric models (−47 Kcal/mol and −36 Kcal/mol, respectively), suggesting that picrotoxin blocks the pores of the ρ2 channels more effectively.     

Increasing the efficiency of silicon and aluminum extraction from Volclay by a water iteration treatment for the synthesis of MCM-41 nanomaterials

This work aims to reduce the prices of a wide range of nanomaterials which are unreachable in the industry by using natural sources as silicon and aluminum precursors. In a previous work, silicon and aluminum have been extracted from Volclay after applying the alkaline fusion process at 550 °C, and a water treatment of this fused clay by adopting a weight ratio (1:4, fusion mass:H₂O) to synthesize Al-MCM-41 nanomaterials. In this study, the weight ratio of fusion mass:H₂O was increased to 1:8 to synthesize a highly structurally ordered MCM-41 under the same reaction conditions. The Al-MCM-41 nanomaterials are investigated by inductively coupled plasma optical emission spectrometry (ICP–OES), powder X-ray diffraction (XRD), N₂ adsorption–desorption measurements and scanning electron microscopy (ESEM). As a result, the increase in the weight ratio fusion mass:H₂O generates more silica and aluminum, which allows the formation of well-ordered MCM-41 nanomaterials with high pore volume (0.70 cm³/g), high surface area (1044 m²/g), and uniform mesoporous diameter (3.67 nm); as a consequence, the increase in the weight ratio fusion mass:H₂O leads to an increase in the mass of Al-MCM-41 (9.3 g for 1:8 compared to 5 g for 1:4), whereas the yield of production of mesoporous materials increases to 86%.     

PAS-cal: a Generic Recombinant Peptide Calibration Standard for Mass Spectrometry

We describe the design, preparation, and mass-spectrometric characterization of a new recombinant peptide calibration standard with uniform biophysical and ionization characteristics for mass spectrometry. “PAS-cal” is an artificial polypeptide concatamer of peptide cassettes with varying lengths, each composed of the three small, chemically stable amino acids Pro, Ala, and Ser, which are interspersed by Arg residues to allow site-specific cleavage with trypsin. PAS-cal is expressed at high yields in Escherichia coli as a Small Ubiquitin-like MOdifier (SUMO) fusion protein, which is easily purified and allows isolation of the PAS-cal moiety after SUMO protease cleavage. Upon subsequent in situ treatment with trypsin, the PAS-cal polypeptide yields a set of four defined homogeneous peptides in the range from 2 to 8 kDa with equal mass spacing. ESI-MS analysis revealed a conveniently interpretable raw spectrum, which after deconvolution resulted in a very simple pattern of four peaks with similar ionization signals. MALDI-MS analysis of a PAS-cal peptide mixture comprising both the intact polypeptide and its tryptic fragments revealed not only the four standard peptides but also the singly and doubly charged states of the intact concatamer as well as di- and trimeric adduct ion species between the peptides, thus augmenting the observable m/z range. The advantageous properties of PAS-cal are most likely a result of the strongly hydrophilic and conformationally disordered PEG-like properties of the PAS sequences. Therefore, PAS-cal offers an inexpensive and versatile recombinant peptide calibration standard for mass spectrometry in protein/peptide bioanalytics and proteomics research, the composition of which may be further adapted to fit individual needs.

Automated design of protecting molecules for metal nanoparticles by combinatorial molecular simulations

New tripod oligo(dibenzyl sulfide) molecules were designed by computer modeling calculations so that they would form 1:1 complexes with an Au₁₄₇ nanoparticle. Twelve aromatic molecules containing two methylthiomethyl groups were used as construction units (“residues”). Combinations of the residues (“sequences”) were examined by molecular dynamic simulations, and those sequences giving the largest interaction energies with the gold nanoparticle were sought through either full search or genetic algorithm. Best-fit sequences were found for N = 5 and 6 (N is the number of “residues” in one leg of the tripod molecule).     

Volumetric and compressibility behaviour of poly(propylene glycol) – Amino acid aqueous solutions at different temperatures

Precise density and sound velocity measurements have been carried out for aqueous solutions of PPG725 in the absence and presence of (0.2 and 0.5) mol · kg⁻¹ amino acids: alanine, glycine, serine and proline, and also for aqueous solutions of these amino acids in the absence and presence of 0.01 w/w PPG725 at T = (288.15, 293.15, 298.15, 303.15 and 308.15) K. From the experimental density and sound velocity values, the apparent molar volume and isentropic compressibility have been obtained and extrapolated to infinite dilution. The infinite dilution apparent molar properties for transfer of PPG from water to aqueous amino acids solutions and also those for transfer of amino acids from water to aqueous PPG solutions have been studied. Temperature dependency of the infinite dilution apparent molar volume was utilised to determine structure-breaker or structure-maker effects of the solutes. Hydration numbers of the amino acids in the investigated aqueous solutions have been evaluated from the volumetric and compressibility properties. All results are discussed based on the salting-out aptitude of the amino acids (hydrophilic + hydrophobic) interactions and (hydrophobic + hydrophobic) interactions occurred between PPG and the investigated amino acids.     

Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3

The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and 252Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17,000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites. Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.     

Sequence-specific predictive chromatography to assist mass spectrometric analysis of asparagine deamidation and aspartate isomerization in peptides

Deamidation of asparagine and spontaneous isomerization of aspartic acid in proteins and peptides occur frequently. These modifications result in a mixture of peptide variants containing all three residues in the sequences. Identification and isomer quantification for these systems are challenging tasks for tandem mass spectrometry commonly utilized in protein analysis. Chromatographic data provide a set of sequence-specific information complementary to mass spectrometry. In order to compare measured retention times (RTs) with those calculated from the sequences derived from protein databases, it is necessary to develop chromatographic models and tools allowing the prediction of RT and elution order for peptides with modified residues. In this work we extended recently introduced critical liquid chromatography of biomacromolecule model for prediction of RTs for peptides containing asparagines, aspartic acid, and isoaspartic acid residues.     

Performance limits and kinetic optimization of parallel and serially connected multi-column systems spanning a wide range of efficiencies for liquid chromatography

Using a set of experimentally determined liquid chromatography column performance data, it has been investigated how a range of efficiencies can best be covered when using a multi-column system. Two main variants are considered: a serially-connected variant (realizing different column lengths by connecting a different number of column segments in series) and a parallel-connected variant (realizing different column lengths by simply switching between columns with a different length arranged in parallel). Both variants are compared for their ability to keep the average analysis time along a given range of efficiencies as close as possible to the intrinsic Knox & Saleem-speed limit. It was found that the serial connection mode offers a better compromise between average speed and amount of required silica (total required column length) than the parallel connection mode for all efficiency ranges running from 5000–10,000 plates up to 75,000–150,000 plates. Considering an ultra-high performance liquid chromatography (UHPLC) operation at 1200 bar, the best possible serial connection system can get within about within 15–25% of the Knox & Saleem-speed limit, whereas a three-column parallel system can only get to within 40–50% of the speed limit, while needing 50–100% more total column length. In absolute terms, the serially-connected system with individually optimized segment lengths should be able to cover a range of 5000–75,000 theoretical plates (dynamic range of 25) in an average analysis time of 14.3 min when using a 1200 bar instrument. At 400 bar, this would be 37.9 min, showing that the construction of wide-efficiency range systems would be one of the application areas where the advantages of UHPLC-conditions would be most fully realized.     

Electrochemical detection of modified maize gene sequences by multiplexed labeling with osmium tetroxide bipyridine

In this report we demonstrate an approach for the electrochemical detection of four sequences from maize and genetically modified (GM) maize by means of square-wave voltammetry (SWV). After multiplexed labeling with osmium tetroxide bipyridine ([OsO₄(bipy)]), the target oligonucleotides are hybridized with a complementary DNA capture probe immobilized on gold electrodes. The multiplexed labeling was performed by mixing the four target strands with the respective oligonucleotides 80% homologous to the central target recognition sequences in order to protect the latter from binding of [OsO₄(bipy)] to its thymine or cytosine residues. All components were added to the same solution. No significant decreases in SWV hybridization signals were observed after such multiplexed labeling of up to four target strands in the same reaction batch. Obtained voltammetric signals were significantly higher at 50 °C compared to 25 °C hybridization temperature and very low response was observed for non-complementary strands. Multiplexed labeling with osmium tetroxide bipyridine holds great promise for the development of simple and effective voltammetric detection protocols for GM organisms.     

Thermodynamics of fusion and sublimation for a homologous series of eleven alkane-α,ω-diols HO-(CH2)n-OH: Structure-related odd–even effect

Temperatures and enthalpies of fusion and solid-to-solid transition of a homologous series of linear alkane-α,ω-diols, HO-(CH₂)_n-OH where n = (6 to 16), were measured by differential scanning calorimetry (d.s.c.). The enthalpies and temperatures of fusion displayed a marked odd–even effect as a function of the number of methylene groups in the alkyl chains, with even terms showing higher values than odd terms. Thermodynamic parameters of fusion were compared with those of isoelectronic linear alkanes and earlier measured alkane-α,ω-diamines, alkane-α,ω-diamides and alkane-α,ω-dinitriles. Results were discussed with reference to the effects of chain length on crystal structures and packing patterns raised on hydrogen bonding and hydrophobic interaction interplay. The enthalpies of sublimation at T = 298.15 K were obtained from the enthalpies of fusion and the literature enthalpies of vaporisation, both adjusted to 298.15 K. A smoother odd–even pattern was observed for the enthalpies of sublimation.     

Degradation of diazinon contaminated waters by ionizing radiation

Study of degradation of diazinon pesticide by ⁶⁰Co gamma irradiation in a single aqueous solution was conducted on a laboratory scale and the effect of ionizing radiation on the removal efficiency of diazinon residues was investigated. Distilled water solutions at three different concentrations of targeted compound (i.e. 0.329, 1.643 and 3.286 μmol dm⁻³) were irradiated over the range 0.1–6 kGy. The initial concentration of contaminant and irradiation doses play a significant role in the rate of destruction; this was evident from the calculated decay constants of diazinon residue. Gamma radiolysis showed that the absorbed doses from 1.5 to 5.6 kGy at a dose rate of 4.79 kGy h⁻¹ achieved 90% destruction for diazinon with initial concentrations over the range 0.329–3.286 μmol dm⁻³. The radiolytic degradation by-products and their mass balances were qualitative determined with good confidence by using GC/quadrupole mass spectrometry (GC/MS) with EI⁺ or CI in positive and negative ionization mode and diazinon degradation pathways were proposed. Additionally, the final products of irradiation were identified by ion chromatography (IC) to be acetic and formic acid.     

Contribution of buried aspartic acid to the stability of the PDZ2 protein

Statistical analysis of protein structures shows that buried aspartic acid residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 potential ionic interactions with other protein groups. To estimate the energetic contribution of such buried groups to the Gibbs free energy of proteins, we measured the effects of amino acid substitutions of D56 in a model protein PDZ2 on its stability. We used temperature-induced unfolding monitored by DSC and denaturant-induced unfolding monitored by the changes in fluorescence intensity. We find that all substitutions of D56 lead to protein unfolding, thus suggesting that this buried hydrogen bonded aspartic acid has a significant contribution to the stability. To quantify the changes in the Gibbs free energy, one of the variants, D56N was stabilized by addition of the protective osmolyte TMAO. Comparison of the stability of the D56N variant with the wild-type PDZ2 in the presence and absence of TMAO allowed us to estimate the contribution of D56 to the protein stability to be 18 kJ · mol⁻¹. These findings are discussed in terms of contribution of buried ionizable groups to protein stability.     

Effects of clustering structure on volumetric properties of amino acids in (DMSO + water) mixtures

For a better understanding on the functions of DMSO in biological systems at a relatively lower concentration, apparent molar volumes of three typical amino acids, glycine, l-alanine and l-serine in (DMSO + water) mixtures were determined and the transfer volumes from water to the mixtures were evaluated. Together with static light scattering measurement, the results were utilised to reveal the microscopic solvent structure of (DMSO + water) mixtures and its influence on the interaction between DMSO and amino acids from a clustering point of view. The results demonstrate that the interaction between amino acids and DMSO is greatly related to the clustering structure of the mixed solvent and that amino acids interacted with already established solvent clusters. The linear dependence of transfer volume of amino acids on DMSO concentration up to 2.0 mol ⋅ dm⁻³ could be attributed to the increasing interaction with (DMSO)₁(H₂O)_n clusters. The formation of (DMSO)_m(H₂O)_n cluster via hydrophobic aggregating at higher DMSO concentration led to a decrease in hydrophobic effect of DMSO and its hydrophobic–hydrophilic and hydrophobic–hydrophobic interaction with amino acids. The structure change of solvent and the interaction between amino acid residues and DMSO was reflected by the solvation of proteins. It was found that dependence of hydrodynamic radius of bovine serum albumin and lysozyme on DMSO concentration was the same and similar to that of static light scattered by the mixed solvent, regardless of the difference in conformational change between the two proteins.     

In vitro digestibility of soybean β-conglycinin hydrolysates

The in vitro digestibility of alcalase enzymatic hydrolysates ofβ-conglycinin was studied.The results showed that the zeta potentials ofβ-conglycinin hydrolysates decreased and their electronegativity increased when digested with pepsin and trypsin.Furthermore,the content of peptides with molecular weight from 10 kDa to 20 kDa remained stable,while those with higher molecular weight(>20 kDa) decreased,and those with lower molecular weight(<10 kDa) increased.The proportion of highly hydrophobic peptides decreased in the process of the in vitro digestion,but no significant change in the surface hydropliobicity indices of digestion products was observed(P<0.05).These results indicate that theβ-congiycinin hydrolysates were degraded through in vitro digestion,but the degree of degradation was relatively low.Peptides with molecular weight from 10 kDa to 20 kDa in theβ-conglycinin hydrolysates resisted the digestion by pepsin and trypsin and they remained stable during the in vitro digestion processes.