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INTRODUCTION A biomarker is measured as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to therapeutic intervention. Many times, a putative biomarker is a protein or peptide [...]

Protein-resistant poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared in 150 [micro]m i.d. capillaries using novel binary porogenic solvents consisting of ethyl ether and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO copolymer with molecular weights (MWs) from 2700 to 5800. The effects of the MWs and concentrations of these surfactants in the porogenic solvent mixture on the pore properties of the resultant monoliths were investigated. Several of the monoliths showed improvements in protein and peptide separations over an extended MW range compared to monoliths synthesized using non-surfactant porogens (i.e., low MW organic liquids). The pore size distributions were examined using inverse size-exclusion chromatography (ISEC) of a select series of proteins and peptides covering a wide MW range. It was found that the best monolith had relatively large fractions of micropores (

Analyses of peptides in complex mixtures are significant challenges in proteomics applications. Here, we report an amphiphilic polymer-based nanoassembly that is capable of selectively extracting peptides, on the basis of their isoelectric points, into an immiscible organic phase from an aqueous solution. The isoelectric point (pI) cutoff in these extractions depends on the pH of the aqueous solution, and thus, sequential fractionation of peptide mixtures based on pI can be accomplished by varying the pH of the aqueous solution. Additionally, we observe an unexpected enhancement in the MALDI-MS signal for extracted peptides ionized in the presence of the polymer, which allows us to obtain reproducible ion signals for some peptides at concentrations as low as 10 pM.

The castor bean plant (Ricinus communis) is used in large quantities for oil production and is also a common ornamental garden plant. However, the beans contain 1-3% of the highly toxic protein ricin, a type II ribosome-inactivating protein that is covered by the Chemical Weapons Convention, and there have been a number of reports concerning the use, or alleged use, of the toxin in terrorist and criminal activities. In the study reported here, we investigated the potential utility of organic solvent-assisted trypsin digestion of crude extracts containing the closely related toxins ricin or abrin to prepare samples for peptide analysis by liquid chromatography combined with electrospray ionization quadrnpole time-of-flight tandem mass spectrometry. Diagnostic tryptic fragments of the toxins were detected and unambiguously identified by this procedure. The sample preparation protocol substantially reduces the sample preparation time, from overnight to an hour, and thus greatly reduces the total time required for analyses, to less than 2 h. Furthermore, the reported procedure leaves the disulfide bonds in the protein intact. This is highly relevant in the context of the Chemical Weapons Convention, since the disulfide bond connecting the two chains of ricin indicates the presence of an intact toxin and provides additional forensic evidence for the analytical results.

Most algorithms for identifying peptides from tandem mass spectra use information only from the final spectrum, ignoring non-mass-based information acquired routinely in liquid chromatography tandem mass spectrometry analyses. One physiochemical property that is always obtained but rarely exploited is peptide chromatographic retention time. Efforts to use chromatographic retention time to improve peptide identification are complicated because of the variability of retention time in different experimental conditions--making retention time calculations nongeneralizable. We show that peptide retention time can be reliably predicted by training and testing a support vector regressor on a small collection of data from a single liquid chromatography run. This model can be used to filter peptide identifications with observed retention time that deviates from predicted retention time. After filtering, positive peptide identifications increase by as much as 50% at a false discovery rate of 3%. We demonstrate that our dynamically trained model generalizes well across diverse chromatography conditions and methods for generating peptides, in particular improving peptide identification using nonspecific proteases.

The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/ TOF mass spectrometry, collecting statistics using acurated set of 2459 MS/MS spectra and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest mass peaks. The amino acids P, W, D, and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K, and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation.

The abundance ratio between the light and heavy isotopologues of an isotopically labeled peptide can be estimated from their selected ion chromatograms. However, quantitative shotgun proteomics measurements yield selected ion chromatograms at highly variable signal-to-noise ratios for tens of thousands of peptides. This challenge calls for algorithms that not only robustly estimate the abundance ratios of different peptides but also rigorously score each abundance ratio for the expected estimation bias and variability. Scoring of the abundance ratios, much like scoring of sequence assignment for tandem mass spectra by peptide identification algorithms, enables filtering of unreliable peptide quantification and use of formal statistical inference in the subsequent protein abundance ratio estimation. In this study, a parallel paired covariance algorithm is used for robust peak detection in selected ion chromatograms. A peak profile is generated for each peptide, which is a scatterplot of ion intensities measured for the two isotopologues within their chromatographic peaks. Principal component analysis of the peak profile is proposed to estimate the peptide abundance ratio and to score the estimation with the signal-to-noise ratio of the peak profile (profile signal-to-noise ratio). We demonstrate that the profile signal-to-noise ratio is inversely correlated with the variability and bias of peptide abundance ratio estimation.

A new method that allows fast and quantitative recovery of hydrophobic or amphipathic peptides, or both, after their intimate incorporation into lipid membranes, is proposed. It relies on the use of small Sep-Pak cartridges and simple chromatographic handling. Peptides selected for this study are the 35 amino acid transmembrane domain of the Neu/erbB-2 protein and its point mutated (V664E) analogue expressed in some cancers, the 25 amino acid BH4 domain from the Bcl-2 antiapoptotic protein and the 15 amino acid Catestatin segment from chromogranin A found to have antimicrobial capabilities. Incorporation of peptides into membranes is accomplished using organic solvent cosolubilization and several cycles of freeze-drying/hydration from aqueous solution. For the hydrophobic peptides, separation from the membrane is performed on Sep-Pak [C.sub.2] columns in two steps: (i) water/methanol elution of lipids and (ii) peptide elution using aprotic solvents (acetonitrile, 2-propanol). For amphipathic peptides, separation is performed on Sep-Pak [C.sub.l8] columns using selective elution in one single step: water/methanol elution to recover first the peptide and then the lipids. Peptide and lipid recovery after all purification steps range from 60 to 80%, with peptide purity above 96%. This new method is simple, inexpensive, and very fast: a 10-mg membranous mixture containing 10% (w/w) peptide may be separated in 20-30 man.

Two-dimensional liquid chromatography is often used to reduce the proteomic sample complexity prior to tandem mass spectrometry analysis. The 2D-LC performance depends on the peak capacity in both chromatographic dimensions, and separation orthogonality. The peak capacity and selectivity of many LC modes for peptides is not well known, and mathematical characterization for orthogonality is underdeveloped. Consequently, it is difficult to estimate the performance of 2D-LC for peptide separation. The goal of this paper was to investigate a selectivity of common LC modes and to identify the 2D-LC systems with a useful orthogonality. A geometric approach for orthogonality description was developed and applied for estimation of a practical peak 2D-LC capacity. Selected LC modes including various RP, SCX, SEC, and HILIC were combined in 2D-LC setups. SCX-RP, HILIC-RP, and RP-RP 2D systems were found to provide suitable orthogonality. The RP-RP system (employing significantly different pH in both RP separation dimensions) had the highest practical peak capacity of 2D-LC systems investigated.

Ion mobility spectrometry (IMS) has been explored for decades, and its versatility in separation and identification of gas-phase ions is well established. Recently, field asymmetric waveform IMS (FAIMS) has been gaining acceptance in similar applications. Coupled to mass spectrometry (MS), both IMS and FAIMS have shown the potential for broad utility in proteomics and other biological analyses. A major attraction of these separations is extremely high speed, exceeding that of condensed-phase alternatives by orders of magnitude. However, modest separation peak capacities have limited the utility of FAIMS and IMS for analyses of complex mixtures. We report 2-D gas-phase separations that join FAIMS to IMS, in conjunction with high-resolution and accuracy time-of-light (TOF) MS. Implementation of FMMS/IMS and IMS/ MS interfaces using electrodynamic ion funnels greatly improves sensitivity. Evaluation of FAIMS/IMS/TOF performance for a protein mixture tryptic digest reveals high orthogonality between FAIMS and IMS dimensions and, hence, the benefit of FMMS filtering prior to IMS/MS. The effective peak capacities in analyses of tryptic peptides are ~500 for FAIMS/IMS separations and ~[10.sup.6] for 3-D FAIMS/IMS/MS, providing a potential platform for ultrahigh-throughput analyses of complex mixtures.

In this study, S. cerevisiae crude membrane fractions were prepared using the acid-labile detergent RapiGest from cells grown under rich and minimal media conditions using [sup.14]N and [sup.15]N ammonium sulfate as the sole nitrogen source. Four independent MudPIT analyses of 1:1 mixtures of sample were prepared and analyzed via quantitative multidimensional protein identification technology on a two-dimensional ion trap mass spectrometer. Using the method described in this study, low-abundance integral membrane proteins with up to 14 transmembrane domains were identified and their protein expression determined when sufficient spectrum counting and ion chromatogram information was generated. We demonstrate that spectrum counting and mass spectrometry derived ion chromatograms strongly correlate for determining quantitative changes in protein expression. Spectrum counting proved more reproducible and has a wider dynamic range contributing to the deviation of the two quantitative approaches from a perfect positive correlation.

A wide variety of mechanisms have evolved for intercellular communication in metazoans, but some of the signaling molecules were already used in their predecessors. The social amoeba, Dictyostelium discoideum, is known to use peptides to trigger sporulation within fruiting bodies, but their sequences have not been defined. We found that the peptide signal spore differentiation factor 2 (SDF-2) is processed from acyl-CoA binding protein, AcbA. The mammalian homolog of AcbA is processed to diazepam binding inhibitor that binds to the GAB[A.sub.A] receptor in the brain and to peripheral 1,4 benzodiazepine receptors. Although Dictyostelium has neither GAB[A.sub.A] nor peripheral-type benzodiazepine receptors, we find that both a diazepam binding inhibitor peptide and diazepam (Valium) can mimic SDF-2 in a Dictyostelium bioassay. Mutants lacking AcbA sporulate well only when developed in chimeras with WT cells. Using a yeast system we show that ligand binding to the SDF-2 receptor histidine kinase, DhkA, inhibits phosphorelay, which can account for its ability to induce rapid sporulation. receptor histidine kinase | diazepam binding inhibitor | triakontatetraneuropeptide | octadecaneuropeptide | diazepam

In this work, we describe the application of a stable isotope amino acid (lysine) labeling in conjunction with data-dependent multiplexed tandem mass spectrometry (MS/MS) to facilitate the characterization and identification of peptides from proteomic (global protein) digests. Lysine auxotrophic yeast was grown in the presence of [sup.13]C-labeled or unlabeled lysine and combined after harvesting in equal proportions. Endoproteinase LysC digestion of the cytosolic fraction produced a global proteomic sample, consisting of heavy/light labeled peptide pairs. Then data-dependent multiplexed-MS/MS was applied to simultaneously select and dissociate only labeled peptide ion pairs. The approach allows differentiation between N-terminal (e.g., b-type ions) and C-terminal fragment ions (e.g., y-type ions) in resulting tandem mass spectra, as well as the capability of differentiation between near-isobaric glutamine and lysine residues. We also describe the utility of peptide composition and fragment information to support peptide identifications and examine the potential application of lysine labeling for differential quantitative protein analysis.

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

A reversed-phase high-performance liquid chromatography (HPLC) separation approach has been combined with ion mobility/time-of-flight (TOF) mass spectrometry in order to characterize a combinatorial peptide library designed to contain 4000 peptides of the general form [NH.sub.2]-Xxx-Xxx-Xxx-[CO.sub.2]H, [NH.sub.2]-Ala-Xxx-Xxx-Xxx-[CO.sub.2]H, [NH.sub.2]-Ser-Ala-Xxx-Xxx-Xxx-[CO.sub.2]H and [NH.sub.2]-Leu-Ser-Ala-Xxx-Xxx-Xxx-[CO.sub.2]H (where Xxx represents a randomization over 10 different amino acids: Ala, Arg, Asp, Glu, Gly, Leu, Lys, Phe, Ser, and Val). Addition of the gas--phase mobility separation between the HPLC separation and TOF measurement dimensions makes it possible to resolve many peptide isomers that have identical retention times (and masses).

Research into the helix-coil transition of a synthetic alpha-helical peptide, Ac-YGG(KAAAA)3-CO-D-Arg-CONH2, is presented. The multiexponential relaxation kinetics indicate that the helix-coil transition is not a two-state process.

Subfemtomole peptide sequence analysis has been achieved using microcapillary HPLC columns, with integrated nanoelectrospray emitters, coupled directly to a Fourier transform ion cyclotron resonance mass spectrometer. Accurate mass (+/- 0.010 Da) peptide maps are generated from a standard six-protein digest mixture, whose principle components span a concentration dynamic range of 1000:1. Iterative searches against approximately 189 000 entries in the OWL database readily identify each protein, with high sequence coverage (20-60%), from as little as 10 amol loaded on-column. In addition, a simple variable-flow HPLC apparatus provides for on-line tandem mass spectrometric analysis of tryptic peptides at the 400-amol level. MS/MS data are searched against approximately 280 000 entries in a nonredundant protein database using SEQUEST. Accurate precursor and product ion mass information readily identifies primary amino acid sequences differing by asparagine vs aspartic acid ((delta)m = 0.98 Da) and glutamine vs lysine ((delta)m = 0.036 Da).

Research demonstrates production of fermented milks containing angiotensin-I-converting-enzyme-inhibitory peptides by two species of Lactobacillus species. Data on ACE-inhibitory peptide sequences, chemical synthesis, and biological activity are presented.

Research into the impact of a neighbouring water or methanol molecule on proton migration in a peptide is presented. Solvent-catalyzed tautomerism may play a significant part in the fragmentation of electrosprayed, protonated peptides in the gas phase.

Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins. Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry. In this work, methods for the high-efficiency capillary isoelectric focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide isoelectric point. Under typical nondenaturing CIEF conditions, peptides are concentrated approximately 500-fold, and peptides present at < 1 ng/ (mu)L were detectable using conventonal UV detection. CIEF separations of peptides provided much faster measurements of isoelectric points compared with conventional isoelectric focusing in gels. Very small differences in peptide isoelectric points ((delta)pI approximately 0.01) could be resolved. High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using capillary zone electrophoresis and packed capillary reversed-phase liquid chromatography.

Results show that the Lactococcus lactis peptide transport protein DtpT is specific for di- and tripeptides and show high affinity to hydrophobic amino acid residues. Data also suggest that bacterial transporter exhibit a restrictive substrate recognition as opposed to eukayotic homologue tranport proteins.

In this paper, we describe a general procedure to evaluate the thermodynamics of the interaction between polypeptides and hydrophobic ligands in the presence of aquo-organic solvent mixtures. These studies address experimental requirements for the determination of the linear free energy relationships, derivation of partition coefficients or other extrathermodynamic parameters such as contact areas, or assessment of the conformational changes that may occur when polypeptides or proteins interact with immobilized nonpolar ligands. Not unexpectedly from thermodynamic arguments, the trends and magnitudes of free energy parameters, such as the enthalpy of association, as previously derived in many studies from gradient elution reversed-phase high-performance liquid chromatographic (RP-HPLC) measurements are often different from the data for the same parameters derived from equilibrium binding or microcalorimetric determinations. To reconcile these divergencies and to more closely examine the thermodynamic basis of the interaction of polypeptides with nonpolar ligands, the dependency of the logarithmic capacity factor, ln (mathematical expression not reproducible in ASCII)', on temperature, T, for several polypeptides (bombesin, (Beta)-endorphin, glucagon) have been investigated using a n-butylsilica and acetonitrile - water or methanol - water mixtures of defined solvent compositions. With low-pH, acetonitrile - water mixtures, the van't Hoffplots, i.e., the plots of ln (mathematical expression not reproducible in ASCII)' versus 1/T, were nonlinear over the range of T = 278-358 K, although within a narrow temperature range, e.g., from T = 278-308 K, the experimental data for these polypeptides could be approximated by a linear relationship. This nonclassical van't Hoff behavior was associated with interactive processes that involved temperature-dependent enthalpic, entropic, and heat capacity changes. In contrast, with low-pH, methanol-water mixtures, the van't Hoff plots showed dependencies that were essentially linear over the range of T = 278-358 K. The slopes of the van't Hoff plots with acetonitrile-water and methanolwater mixtures at a defined T value and solvent composition were significantly larger than those found for the corresponding experiments carried out under gradient elution RP-HPLC conditions. From these plots of ln k' versus 1/T, the changes in the apparent enthalpy of association ((Delta)H(super +/-)(sub assoc)) and the apparent entropy of association ((Delta)S(super +/-)(sub asso)) for the interaction of these polypeptides with the solvated n-butyl ligands at different T and solvent compositions have been determined. For these polypeptides, both (Delta)H(super +/-)(sub assoc) and (Delta)S(super +/-)(sub assoc) exhibited linear dependencies on the volume fraction, (mathematical expression not reproducible in ASCII), of the organic solvent over a narrow range of T, but the slopes of these plots were dependent on the T range examined. The dependencies of the slope term, S, and the intercept term, ln (mathematical expression not reproducible in ASCII)(sub 0), derived from the plots of In (mathematical expression not reproducible in ASCII)' versus (mathematical expression not reproducible in ASCII) as a function of T, have also been investigated. A new relationship linking the S values with(Delta)H(super +/-)(sub assoc) and (Delta)S(super +/-)(sub assoc) as a function of T and (mathematical expression not reproducible in ASCII) has been derived and validated. In addition, the relationship between S, (Delta)H(super +/-)(sub assoc) and (Delta)S(super +/-)(sub assoc), the apparent change in heat capacity, (Delta)C(super +/-)(sub assoc), and the accessible surface area, (Delta)A(sub tot), of these polypeptides has been examined, thus providing a linkage of these thermodynamic and extrathermodynamic parameters to the partition coefficient, P, and the molecular properties of these polypeptides. The results confirm that entropy - enthalpy compensation effects participate in the interaction of polypeptides with hydrophobic ligands. This investigation has confirmed that the use of solvent - water mixtures of defined composition, rather than the more convenient practice of using gradient elution methods, is essential if thermodynamically consistent values of the binding affinities and partition coefficients are to be quantitatively derived. Similar considerations apply to the derivation of extrathermodynamic parameters associated with the conformational transitions of polypeptides (or proteins) when they interact with nonpolar n-alkyl ligands. This study thus provides a general approach to evaluate the interaction thermodynamics of polypeptides or proteins in these and similar lipophilic systems.

High-performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic chromatography of plasmid DNA conformations. However, no study about the separation of small molecules has been performed until now. In this work, we investigated the possibility of gradient and isocratic HPMC of small molecules with Convective Interaction Media disks of different chemistries and tried to explain the mechanism that enables their separation. We demonstrated that it is possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed-phase mode. It was shown that similar peak resolution can be provided in both gradient and isocratic modes.

Certain peptides such as dynorphin A [dynA-(1-13)] enhance the release of antigenic peptides bound to class II MHC molecules at neutral pH. This enhanced release has been termed push off. Previous work has shown that the antigenic pigeon cytochrome c peptide PCC-(89-104) has at least two conformational isomers when bound to the class II MHC protein I-[E.sup.k]. We have accordingly studied the push off of PCC-(89-104) from the complex PCC-(89-104)/I-[E.sup.k] to see whether these isomeric conformations are distinguished by the push-off effect. A comparison of the association and dissociation kinetics of PCC-(89-104)/I-[E.sup.k] in the presence of dynA-(l-13) shows that dynA-(1-13) does not simply replace PCC-(89-104) but rather acts catalytically. The major product is peptide-free I-[E.sup.k], which is receptive to further peptide binding. Evidence is presented that a two peptide - one MHC complex is formed in solution. This ternary complex represents the first step of the mechanism of push off. 19F NMR data are presented that indicate that dynA-(1-13) interacts specifically with only one of the two isomeric complexes of PCC-(89-104) and I-[E.sup.k]. A push-off mechanism is proposed in which dynA-(1-13) binds outside the peptide binding groove. In a second step, the dissociation of one of the two isomers is specifically enhanced. Thus the push-off effect may be useful for identifying conformational isomers and for separating them.

Phytochelatins, a class of posttranslationally synthesized peptides, play a pivotal role in heavy metal, primarily [Cd.sup.2+], tolerance in plants and fungi by chelating these substances and decreasing their free concentrations. Derived from glutathione and related thiois by the action of [Gamma]-glutamylcysteine dipeptidyl transpeptidases (phytochelatin synthases; EC 2.3.2.15), phytochelatins consist of repeating units of [Gamma]-glutamylcysteine followed by a C-terminal Gly, Ser, or [Beta]-Ala residue [poly-[([Gamma]-Glu-Cys).sub.n]-Xaa]. Here we report the suppression cloning of a cDNA (AtPCS1) from Arabidopsis thaliana encoding a 55-kDa soluble protein that enhances heavy-metal tolerance and elicits [Cd.sup.2+]-activated phytochelatiu accumulation when expressed in Saccharomyces cerevisiae. On the basis of these properties and the sufficiency of immunoaffinity-purified epitope-tagged AtPCS1 polypeptide for high rates of [Cd.sup.2+]-activated phytochelatin synthesis from glutathioue in vitro, AtPCS1 is concluded to encode the enzyme phytochelatin synthase.

We report an enthalpic factor involved in determining helix propensities of nonpolar amino acids. Thermal unfolding curves of the five 13-residue peptides, Ac-K[A.sub.4]X[A.sub.4]KGY-N[H.sub.2] (X = Ala, Leu, Ile, Val, Gly), have been measured by using CD in water/trifluoroethanol (TFE) mixtures. The peptide helix contents show that the rank order of helix propensities changes with temperature: although Ala has the highest helix propensity at 0 [degrees] C in all TFE concentrations, it is lower than Leu, lie, and Val at 50 [degrees] C in 20% TFE. This change is attributed to shielding by nonpolar side chains of the interaction between water and polar groups in the helix backbone for the following reasons. (i) Helix content is directly related to helix propensity for these designed peptides because side-chain-side-chain interactions are absent. (ii) The change in rank order with temperature is enthalpic in origin: in water, the apparent enthalpy of helix formation calculated from the thermal unfolding curves varies widely among the five peptides and has the same rank order as the helix propensities at 0 [degrees] C. The rank order does not result from burial of nonpolar surface area because the calculated heat capacity change ([Delta]Cp) on helix formation is opposite in sign from the expected [Delta]Cp. (iii) A nonpolar side chain can exclude water from interacting with helix polar groups, according to calculations of water-accessible surface area, and the polar interaction between water and peptide polar groups is entirely enthalpic, as shown by amide transfer data.

Cleavage of peptide bonds with commercially available salts of (PdCl4)(super 2-) anion is shown for the first time to be possible, with no chemical derivatization or modification required of the complex. However, kinetics histidine-containing peptides can be controlled by the choice of ligands in palladium(II) complexes. While selectivity of enzymes is dependent on the structural fit between the active site and the substrate, selectivity of the new artificial dipeptidases depends on the proximity of the anchored metal compound to the scissile amide bonds.

The stability and properties of alphatalpha and its constituent helices were examined via dissection of the de novo designed peptide alphatalpha. The results suggested very little association of the peptides to form either homo- or heterodimers which could mean that stability of helix association is attributed to the high effective concentration of the helices caused by the presence of the connecting turn. The reflection of the effects of salt on the helicity of the component peptides by the intact molecule imply the importance of short-range interactions for the molecule's conformation.

A method for mass spectrometric peptide mapping was developed, based on hydrolysis of a solid protein by acid vapor followed by mass spectrometric analysis of the cleavage products. The method is applicable to lyophilized samples as well as proteins present in gels after separation by SDS-PAGE. The cleavage specificity was established using a number of standard proteins. Three different types of cleavages were observed: specific internal backbone cleavages at Asp, Ser, Thr, and Gly and N- and C-terminal sequence ladders. On the basis of the observed cleavage characteristics, a strategy for protein identification based on the peptide mass maps was developed. The identification strategy utilizes the specific internal backbone cleavages as well as the partial sequence information, obtained from the sequence ladders.

We report the development of a method to compare collision-induced dissociation (CID) spectra of peptides. This method employs a cross-correlation analysis of a CID spectrum to a reference spectrum and normalizes the cross-correlation score to the autocorrelation of the CID spectra. The query spectrum is compared by using both mass information and fragmentation patterns. Fragmentation patterns are compared to each other using a correlation function. To evaluate the specificity of the approach, a set of 2180 tandem mass spectra obtained from both triple-quadrupole tandem mass spectrometers (TSQ) and quadrupole ion trap mass spectrometers (LCQ) was created. Comparisons are performed between tandem mass spectra obtained on the same instrument type as well as between different instrument types. Accurate and reliable comparisons are demonstrated in both types of analyses. The scores obtained in the cross-comparison of TSQ and LCQ tandem mass spectra of the same peptide are found to be slightly lower than comparisons performed with spectra obtained on the same instrument type. The method appears insensitive to variations in day-to-day performance of the instrument, minor variations in fragment ion abundance, and instrumental differences inherent in the same instrument model. The use of this method of comparison is demonstrated for library searching and subtractive analysis of tandem mass spectra obtained during LC/MS/MS experiments.

Peptide fractions rich in residues from the amino acid cysteine appear to increase non-haem iron absorbtion in meat. This appear to be the cause behind the 'meat factor,' or the reason why cellular animal proteins enhance the bioavailability of non-haem iron, while plant, milk and egg proteins do not., It is well established that the bioavailability of non-haem iron from foods is enhanced by the presence of meat. However, the nature of the promoter in meat has not yet [...]

A study was conducted on the physiological significance of feedback and /or autocatalytic reactions during in vivo prothrombin activation. The findings indicate that autocatalysis of meizothrombin is not essential to thrombin formation during factor Xa-catalyzed prothrombin activation in reaction systems containing purified proteins. Moreover, cleavage at Arg284-Thr285 influences plasma thrombin formation.

Boundary-activated dissociation (BAD) of peptides has been investigated as an alternative to the use of resonant excitation to effect collision-induced dissociation in the quadrupole ion trap. BAD's nonresonant excitation mechanism overcomes a major drawback in resonant excitation, namely, the variation of the resonant excitation frequency as a function of ion space charging. As with resonant excitation, the pulsed introduction of heavy gases (argon, xenon) extends the applicability of BAD when tandem mass spectrometry is performed on peptide ions. The presence of heavy gases during ion activation allows greater internal energy deposition and also enables BAD to be performed at much lower trapping field strengths (lower q values) than previously reported for this technique. This extends the mass range over which product ions can be collected.

Matrix-assisted laser desorption/ionization mass spectrometry with postsource decay was used to generate fragment ions from peptide fragments containing heteropeptides linked together by two disulfide bonds. Postsource decay analysis of these peptide samples generates a series of singly charged fragment ions that, in addition to the peptide sequence ions, provide useful information for assigning disulfide arrangement in highly bridged disulfide-linked peptides. The assignment was made possible by fragmentation at peptide bonds between two Cys residues in a peptide that constitutes the highly bridged fragment, while retaining the disulfide linkage to the other peptide. Fragmentation using other types of instruments, such as quadrupole ion-trap mass spectrometry with collision-induced dissociation, usually did not generate such fragment ions. The data obtained from postsource decay also provide fragment ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds. The present method is a highly sensitive technique which requires no further sample handling and should be complementary to other classical chemical methods. The method proved useful in facilitating the assignment of disulfide structure in tumor necrosis factor binding protein (TNFbp), which contains 162 amino acids and 13 disulfide bonds (Jones, M.; et al. Biochemistry, in press). Postsource decay analysis of large disulfide-containing peptides usually produces no fragmentation but generates a series of high-intensity ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds.

Cytotoxic macrolide glycosides such as aurisides A and B from Dolabella auricularia were isolated by bioassay-guided fractionation and analyzed by nuclear magnetic resonance (NMR) spectroscopy. The cytotoxic macrolide glycosides are composed of a new type of carbon backbone identified as 5,7,13-trihydroxy-3,9-dioxoheptadecanoic acid. Furthermore, aurisides A and B which contain a bromide-substituted conjugated diene moiety are structurally related to acutiphycins.

Circular dichroism, insertion studies and phosphorous-31 magnetic resonance spctra were used to establish the interaction between the alpha-helical conformation and the beta-amyloid peptide membranes. The electrostatic restriction of the charged peptide residues mediated the process of intramolecular hydrogen bond formation as well as the helix formation. It was indicated that the active form of beta-amyloid peptide was not helical.

Fournier transform infrared spectroscopy (FT-IR) has been found to be very useful in investigating the effects of chain length in a series of helix-forming peptides, Ac-W(EAAAR)nA-NH2, with n = 1,3,5 and 7. Linking curve-fitting with the 2D correlation analysis gives a deconvolution of FT-IR spectral peaks through the -4 degrees C to 95 degrees C temperature range. This gives information about the temperature-dependence of the vibrational parameters of the system. Vibrational frequencies were found to be relatively insensitive to chain length increases beyond around 15 residues.

A study was conducted on the cleavage of peptides containing a 4-fluorophenylalanine-Pro bond to determine the conformational specificity of FPhePro bond cleavage by pepsin. The findings indicate a high degree of selectivity of pepsin cleavage for the trans peptide isomer. Moreover, the isomeric selectivity for X-Pro bond cleavage approaches complete specificity for the trans isomer.

Reversed-phase liquid chromatography (LC) using a nonporous silica support has been combined with electrospray (ES) time-of-flight (TOF) mass spectrometry (MS) for the fast separation and mass detection of peptides. Using this LC method, the resolution of a peptide mixture can be completed is less than 35 s. The resulting chromatographic peak widths are less than 1 s wide. Because of the unique nature of a TOF mass analyzer, complete mass spectra can be acquired at a rate which is sufficient to sample these narrow peaks. When compared with conventional LC, the same separation takes nearly 20 min to complete, and the signal-to-noise ratio observed in the total ion chromatogram is dramatically lower due to the influence of increased background noise in the mass spectra. The limit of detection for a low molecular weight peptide, Val-Pro-Leu, was found to be 6 pmol with the total ion chromatogram and 500 fmol with the reconstructed ion chromatogram. A peptide map of horse heart myoglobin, completed in 3.5 min, is shown as an example of the results which can be obtained from combining this fast LC method with fast ES/TOF/MS detection capability.

Pulse-chase studies were utilized to demonstrate the effect of beta-secretase cleavage of beta-amyloid precursor protein (APP) in the endoplasmic reticulum/intermediate compartment of NT2N cells. Results obtained demonstrated that the turnover of intracellular APPbeta was slower than the turnover of newly synthesized APP sub(FL), confirming that APPbeta was produced from APP sub(FL) inside NT2N neurons even before secretion.

Researchers have been able to develop nanosecond laser temperature-jump equipment which can monitor changes in the optical absorption or fluorescence of a sample. This has played a useful role in the study of the kinetics of the helix-coil transition of an alanine-based peptide following laser-induced temperature jump. It was possible to establish that the rate of helix propagation is relatively slow and that the relaxation rate for redistribution of helix lengths is almost temperature independent.

Resolution of greater than 100 000 is routinely achieved by MALDI-FT-ICR, based on measured peak widths. However, the ability to separate peaks that require this resolution is difficult to obtain in practice due to peak coalescence, a result of coupling of the cyclotron motion of ions with similar frequencies. This phenomenon is accentuated for high space charge, high trapping plate voltages, and high mass. Very low trapping plate voltages with properly chosen transient measurement times are shown here to yield ultrahigh-resolution separation of closely spaced peaks in peptide mixtures. Measurements of the isotope peaks for partially deamidated preparations of substance P or partially reduced/partially deamidated Ala-Gly-[[Arg].sup.8]-vasopressin show as many as six isotope peaks at one nominal mass. In one example, the 13C isotope peak was separated from the 15N isotope, a separation that required a resolution in excess of 180 000. Measurements were made with an external source MALDI-FT-ICR mass spectrometer with a 4.7 T magnet. These data suggest the need for high-resolution measurements for the determination of exact masses for peptide mixtures.

Three novel cytotoxic compounds were isolated from deep-water Lamellomorpha strongylata. The extraction of the cytotoxic compounds such as swinholide H, calyculinamide A and B involved repeated extractions from frozen specimens by utilizing methanol followed by the bioassay-guided purification of the extract. Swinholide H was detected in the extract as a cytotoxic metabolite with a molecular formula of C80H136O20. Furthermore, Apehadex LH-20 chromatography produced a mixture of calyculins A, B and E including calcyculins A, B, E and F from the extract.

Electrospray ionization/surface-induced dissociation (ESI/SID) tandem mass spectrometry were performed on 20 singly- and doubly-protonated oligopeptides to investigate their fragmentation in the gas phase. According to the relative positions of the ESI/SID fragmentation efficiency curves, the relative energetics of their dissociation are a function of peptide size, amino acid composition and sequence.

Two synthetic peptides, Ala and Leu, were characterized by Fourier transform infrared spectroscopy, polarized infrared spectroscopy and circular dichroism spectroscopy, with curve-fitting analysis. The Ala peptide had a beta structure in solution whereas the Leu peptide had a helical structure. Only Leu was shown to bind strongly to the lipid bilayer vesicles and it did not precipitate in buffered solutions. The slow kinetics displayed by Leu suggest complex multiple structures.