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Extraction of biotinylated peptides by streptavidin magnetic beads has been directly coupled to the MALDI-TOF mass analysis. The elution of peptides from the beads is achieved by first mixing the beads with the MALDI matrix solution and removing, after a few minutes, the beads with a magnet; then, the matrix solution containing the biotinylated peptide is directly mass analyzed by MALDI. Three examples are presented to show the capabilities of this procedure to detect biotinylated peptides present at very low concentrations in complex mixtures. Detection limits of less than 100 fmol can be achieved. Such a coupling strategy is of great interest to investigate peptide/protein interactions.

Effective analysis of the sequence of peptides using matrix-assisted laser desorption/ionization (MALDI) tandem ion trap mass spectrometry requires efficient mass isolation and the ability to induce extensive sequence-specific fragmentation. The present paper describes a new excitation scheme, which we term red-shifted off-resonance large-amplitude excitation (RSORIAE), that can deposit higher amounts of internal energy in ions than is feasible with conventional resonant excitation. The new method provides an effective means for inducing fragmentation of MALDI-produced peptide ions with m/z values up to 3500. Prior to excitation, it is necessary to isolate ions of interest with high efficiency. We demonstrate that isolation efficiencies of >95% can be achieved by careful design of the rf scan functions used during ion isolation. In particular, sudden transitions in the amplitude of the rf field (from low to high amplitudes) must be avoided. The combined improvements in the efficiency for ion isolation and the efficacy of ion activation make MALDI tandem ion trap mass spectrometry a practical tool for the characterization of proteins with high sensitivity.

The 19 kDa membranes of a complex of tryptic fragments of sodium plus potassium-activated adenosinetriphosphatase having occluded Rubidium (Rb) and bound ouabain are solubilized a non ionic detergent polyoxyethylene 10-laurylether to detect the fragments necessary to preserve Rb occlusion. A monomer soluble complex containing Rb and consisting of cosolubilized fragments of alpha, beta, and gamma subunits is obtained on a sucrose density gradient. The beta subunit is partially split, whereas cation occlusion and ouabain binding are intact. The stability of the complex is discussed.

The cystine-bearing peptide complex, irreversibly sickled cell (ISC) beta-actin tryptic cytine-peptide (KFC-CDVDIR), was prepared, characterized by spectroscopy and chromatography, and identified in the tryptic digest of ISC beta-actin by mass spectrometry. Interestingly, cysteines284 and 373 of the ISC beta-actin are indeed linked by a disulfide bridge. The carboxy end of actin rearranges upon dissociation of the accompanying protein. This results to the solvent orientation of C373 side segment and the predisposal of C373 and C284 portions to the formation of disulfide bond.

Reverse-phase high performance liquid chromatography with alternative mobile phases is used for the sensory evaluation of bitter peptides in cheese. The resolution of the components of mobile phases is good, if acetonitrile and trifluoroacetic acid are substituted with absolute ethanol and food-grade hydrochloric acid, respectively. Corrected retention times, and the average hydrophobicity and molecular weight of amino acids and peptides are correlated to derive a regression model. The regression model is unable to predict the localization of bitter peptides on a separation profile.

Capillary zone electrophoresis (CZE) is known for its high separation efficiency. Optically gated CZE is a form of CZE that allows rapid analyses while high separation efficiency is maintained. Analysis of peptides containing proline with CZE and optically gated CZE gives two peaks for dipeptides and multiple peaks for oligopeptides containing more than one proline. The multiple peaks are believed to be due to the cis and trans forms possible in peptide bonds containing proline. Isomerization between the cis and trans forms takes place at room temperature with a lifetime of seconds to minutes. Resolution of these peaks in CZE is found to be a function of separation efficiency and time of analysis. Rapid analyses with relatively lower separation efficiency give resolution equal to or better than that of longer analyses with much higher efficiency. This confirms the relationship between the multiple peaks and isomerization between the cis and trans forms. To extract kinetic information from the experimental data, a digital simulation is used to model the separation obtained experimentally. The equilibrium constant and rate constants that give the best fit of the simulated data to the experimental data are considered estimates of the actual values for the given experimental conditions.

Coupled-column two dimensional (2D) separation systems offer potentially high peak capacity and are amenable to automation. Reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) are complementary techniques well suited for use in a 2D system. Optically gated CZE is a means of performing rapid CZE analyses. In a 2D system, these rapid CZE analyses enable more frequent sampling of the RPLC separation and thus more freedom in control of the RPLC analysis conditions. Complete 2D separations can be done in the time usually required to do the RPLC analysis alone. With the present system, a complete 2D analysis can be done in under 10 min. Because the peak capacity of a 2D method is inherently high, some of the available peak capacity can be exchanged for speed of analysis. Acceleration of the RPLC elution gradient will decrease analysis time, but RPLC resolution will suffer. However, because it is a 2D system, some of the resolution lost in the RPLC can be regained in the second dimension CZE analysis. Here, the RPLC gradient was done over only 2 rain, but CZE analyses done every 2.5 s restore some of the resolution lost in the rapid RPLC gradient.

Multidimensional separation methods are attractive because of their potentially high peak capacities. Coupled-column systems in particular offer the advantages of online detection and automation. With the development of rapid two-dimensional (2D) analysis, it becomes possible to consider three-dimensional (3D) separation systems. In such a 3D system, effluent from a slow first dimension is repetitively sampled into a rapid 2D system. In the resultant data, each sample component has been subjected to three separative displacements, and the overall peak capacity is the product of that of each of the three dimensions. This paper demonstrates a comprehensive coupled-column 3D analysis of peptides. Size exclusion chromatography (SEC) is used as the first dimension to separate sample components by molecular weight, over an analysis time of several hours. The SEC effluent is repetitively sampled on-line into a rapid 2D reversed phase liquid chromatography/capillary zone electrophoresis (CZE) system with an analysis time of 7 min. Detection of sample zones is done only afer the final CZE separation, by laser-induced fluorescence detection. Analysis data from this system consist of a series of 2D 'slices' of the SEC effluent, which when stacked together give the 3D separation 'volume'.

Cooperative hydrogen-bonds may play a major role in protein folding. Low fractionation factors (phi) for water clusters and various peptide clusters occur in the presence of both cooperative hydrogen-bonded networks and charged interactionsand The correlation between phi and hydrogen bond is nonlinear with a maxima at N-O bond distance of nearly 2.8 angstrom.

A study of DNA cleavage by synthetic peptides in the presence of Ni(II) and oxone reveals that peptides of the nature NH2-Xaa-Xaa-His-CONH2 are capable of selectively modifying the DNA and possibly form site-selective DNA binding and modifying agents. These metallopeptides initiate strand scission through a non-diffusible oxygen at mixed A/T-rich positions. The reaction is inhibited by the minor groove binding drug, distamycin.

The study demonstrates that class II-associated invariant chain peptides (CLIP) can be subdivided into distinct fractional regions. Series-wise removal of the invariant chain in a trans-Golgi compartment renders major histocompatibility complex class II molecules accessible for peptide loading with CLIP as the last fragment. The C-terminal segment of CLIP fragment inhibits self and antigenic peptide binding to human leukocyte antigen molecules.

The interactions of animal venoms with neuronal calcium channels to produce polypeptides are suitable models for the development of therapeutics aiming towards ionophores. The venom of the spider Segestria florentina is used to produce SNX-325, a novel selective calcium channel antagonist peptide. The SNX-325 is not as potent at displacing radioiodinated SNX-111 from rat brain synaptosomal membrane than SNX-111. SNX-325 shows selectivity for calcium currents. The characteristic and methodology of isolating SNX-325 are discussed.

A Caribbean tunicate extract yielded seven new didemnins: didemnins M, N, X and Y, acyclodidemnin A, epididemnin A1 and nordidemnin N. The didemnins have structural variations in the polarity and length of the peptide side chain, Tyr-derived subunit, cyclic structure and stereochemistry of the Hip residue. Cytotoxicity against P388 cells was observed in didemnins M, N, X, Y, A and B, but epididemnin A1 and acyclodidemnin A showed less cytotoxicity.

Two-dimensional 1H NMR spectroscopy establishes the solution structure of a 35 amino acid peptide, PMP-D2. This peptide isolated from Locusta migratoria possesses three disulfide bridges and a core region having a short antiparallel beta-sheet, which is three stranded. Ca2+ currents are blocked by this peptide, which is stable in organic solvents.

We report the application of aerosol matrix-assisted laser desorption ionization (MALDI) to liquid chromatography/mass spectrometry (LC/MS). The aerosol MALDI experiment uses aerosol liquid introduction in conjunction with pulsed UV laser ionization to form ions from large biomolecules in solution. Mass analysis is achieved in a time-of-flight mass spectrometer. In the LC/MALDI-MS experiment, the matrix solution is combined with the column effluent in a mixing tee. LC/MALDI-MS is demonstrated for the separation of bradykinin, gramicidin S, and myoglobin.

Lewis lung carcinoma (3LL)-specific peptides, which exhibit homology to the connexin 37 protein, is isolated and sequenced by repeated fractionation and total acid extraction. The methods use synthetic octamers for the binding of these sequences to H-2K(super b) molecules on RMA-S cells. The process stimulates anti-tumor cytotoxic T lymphocytes.

Pyrrolidone carboxyl peptidases (pcp) are enzymes that cleave n-terminal residues from peptides and proteins and are encoded in the pcp genes of Pseudomonas fluorescens. The enzyme activity functions through histidine and cysteine residual activities. The pcp gene has a reading frame containing 213 amino acids homologous to pcps from other bacteria.

A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer in equilibrium dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

Electrophoretic mobilities and capacity factors for short peptides were determined in micellar electrokinetic systems (MEKC) using UV detection at 210 nm. On the basis of determined capacity ratios, taurodeoxycholic acid was found to be a selective micellar agent for peptides. Structural features other than hydrophobicity were found to be decisive for the distribution of the peptide s to the micellar phase. Basic peptides of enkephalin-type containing arginine were more highly distributed to the micelles than others, indicating the importance of electrostatic forces in the distribution mechanism. In systems with polyacrylamide-coated capillaries, the micellar velocity toward the anode is larger than the electroosmosis, and conditions permitting splitting of a series of related peptides, i.e., some peptides migrating with the micelles and others toward the cathode resulting in infinite selectivities, were developed. Separation by micellar electrokinetic chromatography is demonstrated to be a suitable alternative for peptides that are difficult to separate by capillary zone electrophoresis.

Conventional protein identification and characterization methods are limited by the need for large quantities of sample, incomplete or multiple tagging and the risk of contamination. Consequently, a new method for the separation and identification of peptide fragments digested in the same column is proposed. Digestion of femtomole amounts of beta-lactoglobulin in the column is accomplished by using pepsin. Separation and detection are done using both capillary zone electrophoresis and laser-induced native fluorescence detection.

Selected applications of analytical techniques for the separation and analysis of peptides and proteins are reviewed. This involves the utilization of high-performance liquid chromatography, affinity chromatography, capillary electrophoresis, mass spectrometry and nuclear magnetic resonance (NMR). A separate section on the coupling of mass spectrometry to a separation method and an overview of recent developments in NMR analysis are included.

Collisionally activated dissociation (CAD) of the protonated polyalanines Ala-Ala, Ala-Ala-Ala, and Ala-Ala-Ala-Ala causes breakup of the peptide bonds leading to sequence-indicative fragment ions. The neutral molecules eliminated during these reactions are identified here using neutralization-reionization mass spectrometry (NRMS). N-terminal acylium ions (b(sub n)) arise after the C-terminus is lost as an intact amino acid or peptide; further loss of CO leads to immonium ions (a(sub n)). Upon generation of C-terminal sequence ions (y(sub n)), a hydrogen atom attached to a nitrogen rearranges from the N-terminal to the C-terminal side yielding a protonated amino acid (y(sub 1)) or peptide (y (sub greater than or equal to 2)) as the ionic fragment; the complementary neutral fragment is an aziridinone if the N-terminal amino acid is cleaved and a diketopiperazine if two N-terminal amino acid units are eliminated. Detection of neutral dissociation products can reveal valuable structure information, as demonstrated with the tetrapeptides Val-Gly-Ser-Glu and Val-Gly-Asp-Glu.

The rates of trypsin and chymotrypsin proteolytic action on angiotensin II, (Arg8)vasopressin and (deamino-Cys1,D-Arg8)vasopressin were measured using reversed-phase high-performance liquid chromatography (HPLC) with UV absorbance and flow injection continuous-flow fast atom bombardment (FAB) mass spectrometry (MS) with selected ion monitoring. The results indicated that continuous-flow FAB MS provided rapid detection without relying on chromatographic resolution. However, HPLC featured availability and comparatively inexpensive digestive enzyme detection.

The use of continuousflow fast atom bombardment (CF-FAB) to ionize the sample for tandem mass spectrometry (MS/MS) reduces the background ion current in the sequencing of peptides at the picomole level, thus improving the signal/noise ratio 50 to 100times. The required amount of analyte drops by 25-75%. When paired with array detection (AD), the method registers a detection limit of 900 fmol, well below that of the sequential Edman method. Moreover, CF-FAB-AD-MS/MS can analyze peptide samples at levels where conventional FAB methods cannot yield detectable ions.

Direct control of the zeta potential and electroosmotic flow with an external electric field allows efficient separation and resolution of peptides in capillary zone electrophoresis (CZE). Electroosmotic flow controls migration rates and zone resolutions. The strength and polarity of the zeta potential shape the degree of peptide and protein adsorption via electrostatic interactions. The drop in protein and peptide adsorption at the capillary wall raises the efficiency of their separation. The effects of direct electroosmotic control are studied at various solution conditions.

Peptide mapping of complex proteins at the picomole level requires three key initial steps. The protein is denatured or unfolded to bare cleavage sites. Either reduction-alkylation by tributylphosphine and 2- methylaziridine or oxidation by performic acid then breaks the disulfide bonds.Immobilized trypsin or chymotrypsin or cyanogen bromide finally digests the protein into peptides. Separation is through capillary electrophoresis. Ultraviolet absorbance, enhanced by selective derivatization of arginine- bearing peptides with laser-induced fluorescence, is the detection method of choice. Various mapping methods as well as the problem of reproducibility are discussed.

The Caribbean marine sponge Discodermia sp. yields polydiscamide A, a discodermin depsipeptide that inhibits the growth of the human lung cancer A549. Spectroscopic, chemical degradation and analogue studies reveal a structure composed of 13 amino acids, including a unique 3-methylisoleucine. The side-chain phenylalanine is replaced by p-bromophenylalanine. The lactone ring has five instead of six amino acids. Valine and proline replace the leucine, threonine and sarcosine in the ring. Except for asparagine, which exhibits a D configuration, the common amino acids show the same absolute configuration as in other discodermins.

A high-resolution mass spectrometry was demonstrated to enhanced characterization of large peptides together with the impurities and adduct ions. A number of multiply-charged species of the same mass were generated by electrospray ionization and direct charge designation from the unit mass spacing of isotopes were supplied from isotopic peak resolution. This technique favored its use with tandem mass spectrometry and in amino acid sequences.

Structural studies of the anatialgal tetrapeptide kasumigamide by two-dimensional (sup)1H-(sup)1H and (sup)1H-(sup)13C NMR are presented. Data indicate that kasumigamide is a linear tetrapeptide containing an N-terminal alpha-hydroxy acid and exhibits an antialgal activity against Chlamydomonas neglecta.

This review focuses on selected applications of the separation and analysis of peptides and proteins published during the period of 1997-1998. Specific topic areas covered include high-performance liquid chromatography (HPLC), ultrafiltration, capillary electrophoresis (CE), affinity-based methods for protein isolation and separation, mass spectrometry (MS), detection of nonenzymatic posttranslational modifications, nuclear magnetic resonance spectroscopy (NMR), infrared (IR) and Raman spectroscopy, circular dichroism (CD), UV-visible absorption spectroscopy, dynamic light scattering, and calorimetry. The quantification and identification of peptides and proteins by chromatographic methods and MS have become fairly routine, as has the conformational analysis of peptides and small proteins in solution by CD, IR, and NMR. Therefore, these topics are not reviewed in detail here. In this review, we have attempted to highlight new technological developments or unique applications of analytical methods that impact the analysis of peptides and proteins.

The characterization of a cytolytic peptide from the venom of Conus mustelinus, conolysin-Mt associated with conopeptide mechanisms for destruction of cellular membranes is reported. The newly found 23AA conopeptide, conolysin-Mt is found to have hemolytic activity when tested on human erythrocytes.

Correct identification of proteins from peptide fragments is important for proteomic analyses. Peptides are initially separated by Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) before Mass Spectrometry (MS) identification. At the present time, peptide fragment retention (separation) time is not used as a useful scoring filter for identification of the peptide fragments and their parent proteins. In the present paper, we present a new web-based tool for the prediction of peptide fragment retention times and its use in compiling a database of ∼133,000 peptide fragments computationally obtained by digestion with trypsin of 4,265 E. coli K12 proteins. The retention calculation is based on the described formulae and the fragments/protein identification was carried out using a simple search-scoring algorithm.

Several experimental studies were conducted to demonstrate preferential interaction of the fluoro alcohol component of a fluoro alcohol-water mixture with peptide solutes. The results reveal that the peptide is selectively solvated by hexafluoro-2-propanol to the extent that the fluoro alcohol concentration near the peptide might be 3 to 4 times higher than the nominal concentration of fluoro alcohol in the bulk sample.

A new conceptual basis for the separation of multicomponent molecular mixtures is proposed. A separation method where different components of the mixtures are driven in opposite directions is realized by a judicious combination of two effects, viz., levitation and blow torch effects and a separation factor of 10(super 8) is obtained on nano length scales as compared to 10(super 3), obtainable through conventional methods of separation on macrolength scales.

A one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging and fluorescent bead-sorting instrumentation is utilized to develop a novel, high-throughput method for determining deacetylase substrate specificity. In-solution deacetylase assays on randomly chosen hit and nonhit sequences have shown that hits correlated with increased catalytic activity by as much as 20-fold and SIRT1 can discriminate peptide substrates in a context-dependent fashion.

A description of the high-temperature ultrafast HPLC conditions, along with the instrument modifications needed to reduce the analysis time of each complete second-dimension gradient separation to tens of seconds, is discussed. The dramatic improvement in the speed of reversed-phase gradient elution through the use of high-temperature HPLC and instrument modifications leads to a significant improvement in the performance of two-dimensional HPLC (2D-LC).

Glycoproteins are a functionally important class of biomolecules for which structural elucidation presents a challenge. Fragmentation of N-glycosylated peptides, employing collisionally activated dissociation, typically yields product ions that result from dissociation at glycosidic bonds, with little occurrence of dissociation at peptide backbone sites. We have applied two dissociation techniques, electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD), in a 7-T Fourier transform ion cyclotron resonance mass spectrometer, in the investigation of an N-glycosylated peptide from an unfractionated tryptic digest of the lectin of the coral tree, Erythrina corallodendron. ECD provided c and z ions derived from the peptide backbone, with no observed loss of sugars. Cleavage at 11 of 15 backbone amine bonds was observed. The lack of cleavage at sites located close to the glycosylated asparagine residue may result from steric blocking by the glycan. IRMPD provided abundant fragment ions, primarily through dissociation at glycosidic linkages. The monosaccharide composition and the presence of three glycan branch sites could be determined from the IRMPD fragments. The two types of spectra, obtained with the same instrument, thus provide complementary structural information about the glycopeptide. The current result extends the applicability of ECD for glycopeptide analysis to N-glycosylated peptides and to peptides containing branched, highly substituted glycans.

Pathogenic enterococci are becoming resistant to currently available antibiotics, including vancomycin, the drug of last resort for Gram-positive infections. Enterococci pose a significant public health threat, not least because of the risk of transferring vancomycin resistance to the ubiquitous Staphylococcus aureus. Vancomycin resistance is manifested by cell wall peptidoglycan precursors with altered termini that cannot bind the antibiotic. Small molecules with well-oriented nucleophile-electrophile assembly and complementary chirality to the peptidoglycan termini were identified as catalytic and selective cleavers of the peptidoglycan precursor depsipeptide. These molecules were tested in combination with vancomycin and were found to re-sensitize vancomycin-resistant bacteria to the antibiotic., The first antibiotic-resistant strain of Staphylococcus aureus was identified even before the commercialization of penicillin (1). Over the past decade, the incidence of staphylococcal strains resistant to virtually all antibiotics [...]

A new method for proteolytic stable isotope labeling is introduced to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools or their subfractions. Two [sup.18]O atoms are incorporated universally into the carboxyl termini of all tryptic peptides during the proteolytic cleavage of all proteins in the first pool. Proteins in the second pool are cleaved analogously with the carboxyl termini of the resulting peptides containing two [sup.16]O atoms (i.e., no labeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotope ratios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry. Short sequences and/or accurate mass measurements combined with proteomics software tools allow the peptides to be related to the precursor proteins from which they are derived. Relative signal intensities of paired peptides quantify the expression levels of their precursor proteins from proteome pools to be compared, using an equation described in the paper. Observation of individual (unpaired) peptides is mainly interpreted as differential modification or sequence variation for the protein from the respective proteome pool. The method is evaluated here in a comparison of virion proteins for two serotypes (Ad5 and Ad2) of adenovirus, taking advantage of information already available about protein sequences and concentrations. In general, proteolytic [sup.18]O labeling enables a shotgun approach for proteomic studies with quantitation capability and is proposed as a useful tool for comparative proteomic studies of very complex protein mixtures.

An integrated platform is presented for rapid and sensitive protein identification by on-line protein digestion and analysis of digested proteins using electrospray ionization mass spectrometry or transient capillary isotachophoresis/capillary zone electrophoresis with mass spectrometry detection. A miniaturized membrane reactor is constructed by fabricating the microfluidic channels on a poly(dimethylsiloxane) substrate and coupling the microfluidics to a poly(vinylidene fluoride) porous membrane with the adsorbed trypsin. On the basis of he large surface area-to-volume ratio of porous membrane media, adsorbed trypsin onto the poly(vinylidene fluoride) membrane is employed for achieving ultrahigh catalytic turnover. The extent of protein digestion in a miniaturized membrane reactor can be directly controlled by the residence time of protein analytes inside the trypsin-adsorbed membrane, the reaction temperature, and the protein concentration. The resulting peptide mixtures can either be directly analyzed using electrospray ionization mass spectrometry or further concentrated and resolved by electrophoretic separations prior to the mass spectrometric analysis. This microfluidic system enables rapid identification of proteins in minutes instead of hours, consumes very little sample (nanogram or less), and provides on-line interface with upstream protein separation schemes for the analysis of complex protein mixtures such as cell lysates.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.4-0.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

Baseline resolution of two peptides, RVMRGMR and RSHRGHR, of neutral monoisotopic mass, ~904 Da, has been achieved by microelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at a mass resolving power of ~3 300 000. The elemental compositions of these molecules differ by [N.sub.4]O vs [S.sub.2][H.sub.8] (0.000 45 Da), which is less than one electron's mass (0.000 55 Da)! This result establishes a new record for the smallest resolved mass difference between any two molecules. This achievement is made possible by a combination of high magnetic field (9.4 T), large-diameter (4-in.) Penning trap, and low ion density. The implications for proteomics based on accurate mass measurements are discussed briefly.

A microfabricated fluidic device that combines micellar electrokinetic chromatography and high-speed open-channel electrophoresis on a single structure for the rapid automated two-dimensional analysis of peptides has been devised and demonstrated. The microchip operates by rapidly sampling and analyzing effluent in the second dimension from the first dimension. Second-dimension analyses are performed and completed every few seconds, with total analysis times of less than 10 min for tryptic peptides. The peak capacity of the two-dimensional separations has been estimated to be in the 500-1000 range. The orthogonality of the separation techniques, an important factor for maximizing peak capacity or resolution elements, was verified by examining each technique independently for peptide separations. The two-dimensional separation strategy was found to greatly increase the resolving power over that obtained for either dimension alone.

The continuous gas-phase ion separation and atmospheric pressure focusing properties of high-field asymmetric waveform ion mobility spectrometry (FAIMS) offer significant advantages for the mass spectrometric analysis of tryptic digests of proteins. In this study, tryptic peptides of pig hemoglobin were examined by ESI-FAIMS-MS using a newly designed FAIMS device. The new, hemispherical geometry of the inner electrode served to deliver the ions, via the gas flows, to the center axis of the FAIMS analyzer, improving the sensitivity relative to previous prototypes. Mass spectra collected using this new FAIMS showed significantly less chemical background noise than conventional ESI-MS, while maintaining approximately the same absolute sensitivity as that observed with ESI-MS. As a consequence of the ion separation in FAIMS, the identification of the tryptic fragments was simplified and some peptides, such as the triply protonated [VVAGVANALAHK.sup.3+], that were obscured by the intense background of ESI-MS, were readily detected using ESI-FAIMS-MS. In addition, the FAIMS device was shown to separate isobaric ions at m/z 532.4. Correlations between CV and mass-to-charge ratio, as well as CV and ionic collision cross section, were evaluated for 38 peptide ions identified in the tryptic digest. The correlation between the CV of the peptide and the mass-to-charge ratio is very poor, indicating good orthogonality between the separation by FAIMS and the separation by mass spectrometry.

The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly., The recent explosion of structural information on the bacterial ribosome has set the stage for detailed models explaining both the function and the assembly of this large ribonucleoprotein (RNP) that [...]

Comparison of experimental and predicted S2 values allows one to cross-validate local contact properties of the 3D structure and to assess the state of the protein. The contact strength of neighboring atoms to the peptide plane is a powerful indicator for the amount of ns and sub-ns time scale reorientational N-H motion in globular proteins.

It has been possible to identify a new loop-truncated form of a beta-beta-alpha peptide that creates a well-defined homotrimer in water. The removal of two amino acids in the loop region of the monomer was vital to switching the motif to a discrete oligomeric state.

In this article an instructional procedure to analyze composition and sequence of dipeptides using phenylthiohydantoin amino acid derivaties and high-performance liquid chromaatography (HPLC) is described. In this exercise students learn principles and practical application of HPLC and organic chemistry synthetic techniques.