Separation Of Peptides Research Articles

Preparation and characterization of monodisperse large-porous silica microspheres as the matrix for protein separation

High performance liquid chromatography (HPLC) is a kind of efficient separation technology and has been used widely in many fields. Micro-sized porous silica microspheres as the most popular matrix have been used for fast separation and analysis in HPLC. In this paper, the monodisperse large-porous silica microspheres with controllable size and structure were successfully synthesized with polymer microspheres as the templates and characterized. First, the poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) microspheres (PGMA-EDMA) were functionalized with tetraethylenepentamine (TEPA) to generate amino groups which act as a catalyst in hydrolysis of tetraethyl orthosilicate (TEOS) to form Si-containing low molecular weight species. Then the low molecular weight species diffused into the functionalized PGMA-EDMA microspheres by induction force of the amino groups to form polymer/silica hybrid microspheres. Finally, the organic polymer templates were removed by calcination, and the large-porous silica microspheres were obtained. The compositions, morphology, size distribution, specific surface area and pore size distribution of the porous silica microspheres were characterized by infrared analyzer, scanning-electron microscopy, dynamic laser scattering, the mercury intrusion method and thermal gravimetric analysis, respectively. The results show that the agglomeration of the hybrid microspheres can be overcome when the templates were functionalized with TEPA as amination reagent, and the yield of 95.7% of the monodisperse large-porous silica microspheres can be achieved with high concentration of polymer templates. The resulting large-porous silica microspheres were modified with octadecyltrichlorosilane (ODS) and the chromatographic evaluation was performed by separating the proteins and the digest of BSA. The baseline separation of seven kinds of protein standards was achieved, and the column delivered a better performance when separating BSA digests comparing with the commercial one currently available. The high column efficiency and good reproducibility present that the large-porous silica microspheres obtained can be used as a matrix for peptide and protein separation.

Direct injection of HILIC fractions on the reversed-phase trap column improves protein identification rates for salivary proteins.

Online combination of hydrophilic interaction chromatography (HILIC) and RP chromatography for separation of tryptic peptides is a challenging approach due to the incompatibility of direct loading HILIC fractions on the RP trapping column. High amounts of organic modifiers in loading solvents decrease the binding efficiency of tryptic peptides on C18 phases and lower the number of identifications. A 500 μL loop upfront of the trapping column filled with aqueous mobile phase was employed as a mixing chamber and enabled direct injections and improved saliva protein identification rates of HILIC fractions.

Advances in Technology of Countercurrent Chromatography for Separation of Protein and Peptide

Abstract Countercurrent chromatography is a kind of continuous and effective liquid-liquid partition chromatography with many unique characteristics such as large load capacity, no irreversible adsorption, high recovery rate, low risk of sample denaturation and so on, which has irreplaceable advantages in the separation of proteins and peptides. This review presents the advances of several kinds of new technology of countercurrent chromatography in the separation of proteins and peptides. The development prospect of this field is also discussed.

Now, More Than Ever, Proteomics Needs Better Chromatography

From plant research to biomedicine, proteome analysis plays a critical role in many areas of biological inquiry. Steady improvement in mass spectrometer (MS) technology has transformed the speed and depth of proteome analysis. Proteomes of simple organisms can now be sequenced to near completion in just over an hour. Comparable coverage of mammalian proteomes, however, still requires hours or even days of analysis. Here we ask why current technology fails to achieve comprehensive and rapid analysis of the more complex mammalian proteomes. We propose that further advancements in MS technology alone are unlikely to solve this problem and suggest that concomitant improvements in peptide separation technology will be critical.

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry.

BackgroundThe spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection.Methodology/Principal FindingsTo better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance.ConclusionsThis is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.

Selective extraction of endogenous peptides from human serum with magnetic mesoporous carbon composites

Highly sensitive and selective enrichment of endogenous peptides or proteins from complex bio-system takes a significant important place to the proteomic. In this work, a unique Fe3O4@2SiO2@mSiO2-C nanomaterial was synthesized, contributing to the separation and enrichment of low concentration peptides from complex mixture. The highly ordered mesoporous carbon structure render the nanospheres with unique properties of strongly connected pore channels, strong hydrophobic properties, high specific surface area (254.90m2/g), uniform pore size (3.61nm). Which made it a promising candidates for the efficient enrichment of peptides through hydrophobic-hydrophobic interaction with low detection limit (0.2fmol), superb size-exclusion of high molecular weight proteins, highly selectivity for BSA digest (molar ratio of BSA tryptic digests/BSA, 1:400), ideal peptides recovery (about 87.5%), wonderful repeatability (RSD less than 25%). Moreover, the as-prepared Fe3O4@2SiO2@mSiO2-C nanoparticles were successfully enriched 2198 endogenous peptides from human serum, which fully indicated that the mesoporous carbon nanoparticles was a promising candidate for isolating proteins or peptides from complex biologicals.

Molecular Dynamics Simulations of Amyloid β-Peptide (1-42): Tetramer Formation and Membrane Interactions

The aggregation cascade and peptide-membrane interactions of the amyloid β-peptide (Aβ) have been implicated as toxic events in the development and progression of Alzheimer’s disease. Aβ42 forms oligomers and ultimately plaques, and it has been hypothesized that these oligomeric species are the main toxic species contributing to neuronal cell death. To better understand oligomerization events and subsequent oligomer-membrane interactions of Aβ42, we performed atomistic molecular-dynamics (MD) simulations to characterize both interpeptide interactions and perturbation of model membranes by the peptides. MD simulations were utilized to first show the formation of a tetramer unit by four separate Aβ42 peptides. Aβ42 tetramers adopted an oblate ellipsoid shape and showed a significant increase in β-strand formation in the final tetramer unit relative to the monomers, indicative of on-pathway events for fibril formation. The Aβ42 tetramer unit that formed in the initial simulations was used in subsequent MD simulations in the presence of a pure POPC or cholesterol-rich raft model membrane. Tetramer-membrane simulations resulted in elongation of the tetramer in the presence of both model membranes, with tetramer-raft interactions giving rise to the rearrangement of key hydrophobic regions in the tetramer and the formation of a more rod-like structure indicative of a fibril-seeding aggregate. Membrane perturbation by the tetramer was manifested in the form of more ordered, rigid membranes, with the pure POPC being affected to a greater extent than the raft membrane. These results provide critical atomistic insight into the aggregation pathway of Aβ42 and a putative toxic mechanism in the pathogenesis of Alzheimer’s disease.

Surface-Bound Cucurbit[8]uril Catenanes on Magnetic Nanoparticles Exhibiting Molecular Recognition.

We demonstrate the preparation of surface-bound cucurbit[8]uril (CB[8]) catenanes on silica nanoparticles (NPs), where CB[8] was employed as a tethered supramolecular "handcuff" to selectively capture target guest molecules. In this catenane, CB[8] was threaded onto a methyl viologen (MV(2+) ) axle and immobilized onto silica NPs. The formation of CB[8] catenanes on NPs were confirmed by UV/Vis titration experiments and lithographic characterization, demonstrating a high density of CB[8] on the silica NPs surface, 0.56 nm(-2) . This CB[8] catenane system exhibits specific molecular recognition towards certain aromatic molecules such as perylene bis(diimide), naphthol and aromatic amino acids, and thus it can act as a nanoscale molecular receptor for target guests. Furthermore, we also demonstrate its use as an efficient and recyclable nano-platform for peptide separation. By embedding magnetic NPs inside silica NPs, separation could be achieved by simply applying an external magnetic field. Moreover, the peptides captured by the catenanes could be released by reversible single-electron reduction of MV(2+) . The entire process demonstrated high recoverability.

Towards an automated analysis of bacterial peptidoglycan structure

Peptidoglycan (PG) is an essential component of the bacterial cell envelope. This macromolecule consists of glycan chains alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides containing nonstandard amino acids. Structural analysis of PG usually involves enzymatic digestion of glycan strands and separation of disaccharide peptides by reversed-phase HPLC followed by collection of individual peaks for MALDI-TOF and/or tandem mass spectrometry. Here, we report a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural analysis of PG using high-resolution mass spectrometry and automated analysis of HCD and ETD fragmentation spectra with the Byonic software. Using the PG of the nosocomial pathogen Clostridium difficile as a proof of concept, we show that this high-throughput approach allows the identification of all PG monomers and dimers previously described, leaving only disambiguation of 3–3 and 4–3 cross-linking as a manual step. Our analysis confirms previous findings that C. difficile peptidoglycans include mainly deacetylated N-acetylglucosamine residues and 3–3 cross-links. The analysis also revealed a number of low abundance muropeptides with peptide sequences not previously reported.Graphical The bacterial cell envelope includes plasma membrane, peptidoglycan, and surface layer. Peptidoglycan is unique to bacteria and the target of the most important antibiotics; here it is analyzed by mass spectrometry.

Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient.

Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques.

Separation of Peptides on HALO 2-Micron Particles.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP-HPLC stationary-phase materials with enhanced separation efficiency. HALO packing materials began the revolution in "core-shell" technology with the advantages of faster separations, higher resolution and peak capacity, high temperature stability, and rugged reliable performance compared to traditional HPLC and UHPLC. These materials are characterized by a solid core surrounded by a thin layer of porous material, and represent a technology for the future with continuing refinements. Such refinements are aided via the use of designed synthetic peptide standards during stationary-phase development. Concomitantly, such standards also enable the researcher to monitor RP-HPLC column performance and develop optimized separation protocols for peptides from a wide array of sources. © 2016 by John Wiley & Sons, Inc.

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level.

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical-stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.

DEVELOPMENT OF THE NEW METHOD OF THE MELTED CHEESE PRODUCTS WITHOUT SALT-MELTERS USING CRYOMECHANOLYSIS

The aim of the work is elaboration of the principally new method of deep processing of rennet cheeses to the melting using the complex action of freezing and cryomechanolysis on the raw material that gives a possibility to destruct the hardly soluble biopolymers and to transform them into soluble form. The principally new method of the deep processing of rennet cheeses for receiving the melt cheese products without salts-smelters was elaborated. It differs from the traditional ones by the complete exclusion of the salts-smelters. This method is based on the use of the influence of freezing and fine-dispersed comminution on the raw material. It allows open biological potential of the rennet cheeses more fully and to extract the hidden (bound) protein forms from nanocomplexes of lipids and mineral substances. It allows destruct the proteins of rennet cheeses to the separate polymers and dipeptides and tripeptides. The used technological methods gave a possibility to exclude the salts-smelters at the rennet cheeses manufacturing. They favor the transformation of lipid-proteins paracaseinate calcium phosphate complexes to the separate amino acids and peptides and allow receive homogenous plastic mass. It was established, that at the complex action of freezing and fine-dispersed comminution on the rennet cheese the destruction of hardly soluble lipid-protein nanocomplexes and release of protein from the bound state into free one – nanoform (by 33,5…35 % more) takes place. The mechanisms of this process, connected with cryomechanodestruction of connections between lipids and proteins and non-fermented catalysis of nanocomplexes were described. It was established, that cryomechanodestruction and non-fermented catalysis of protein to the separate monomers – α-amino acids (by 55…60 %) takes place at freezing and fine-dispersed comminution of rennet cheese before melting. The mechanism of freezing and non-fermented analysis, connected with cryomechanocracking of protein molecules at the expanse of peptide protein connections destruction to the separate α-amino acids and their transformation into the free form was described. It was also demonstrated, that the conformational changes of protein molecules take place synchronously. The offered and elaborated nanotechnology of melt cheese products on the base of rennet cheeses without salts-smelters includes complex action of freezing and fine-dispersed comminution. The mechanisms of processes, connected with cryomechanodestruction of connections between lipids and protein to the separate α-amino acids are described. The cheese fillings for “Pancake” confectionary and cheese snacks – falafels were manufactured on the base of cheese mass, received using the new method and enriching vegetable nanoadditives. They exceed the well-known analogs by chemical composition and are remarkable for the storage life, increased in 2 times. At the same time the significant part of substances (BAS and biopolymers) in cheese filings is in nanodimensional form (55…60 % of protein), especially, free α-amino acids, easily assimilated by the human organism. The recipes and technologies of sauces-dressings, sauces-deeps, cheese snacks and so on are also elaborated on the base of cheese mass, received by the new method.

High Efficiency Robust Open Tubular Capillary Electrochromatography Column for the Separation of Peptides

High Efficiency Robust Open Tubular Capillary Electrochromatography Column for the Separation of Peptides

Abstract 3871: Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics

Abstract The dog serves as a useful model of spontaneously occurring cancer by virtue of similar biology, therapies and mechansims of chemotherapeutic resistance. The ABC transporters are a superfamily of membrane bound proteins that are widely distributed across mammalian tissues and constitute a substantial component of variability in anticancer drug pharmakokinetics and tumor cell resistance. Thus a quantitative atlas of ATP-binding cassette (ABC) transporters across canine tissues is crucial for improving our ability to extrapolate data from dog to human. We have employed a quantitative proteomic approach based on multiple reaction monitoring in tandem mass spectrometry that relies on detection of novel peptide sequences following trypsin digestion of biological samples and separation of peptides utilizing high-pressure liquid chromatography. The 14 proteins evaluated include P-gp, Bcrp, Bsep, Mrp1-6, Mrp7, Mrp9, and the products of canine Abcg5 and Abcg8. Peptide sequences were identified via an in-silico trypsin digestion of proteins and signature peptides were generated for identification and quantitation using stable isotope-labeled signature peptides as an internal reference. Signature peptides were chosen based on lack of posttranslational modification sites and known polymorphisms. Isolated brain microvessels, liver, kidney, duodenum, jejunum, ileum and lung were collected at necropsy from ten normal dogs and membrane fractions of each tissue were prepared. Additional samples were collected for isolation of mRNA. Membrane protein fractions were digested with trypsin and total protein concentrations determined by BCA assay. Calibration curves for signature peptides were prepared in a bovine serum albumin digestate and ranged from 0.25 fmol to 200 fmol on column. Quantitation of peptides in tissue samples was performed via linear regression analysis of calibration curves with extrapolation using at least two transitions for each peptide. Results were obtained as femtomole of peptide per microgram of total protein in the sample. Differential protein levels across tissues were compared to quantitative real time RT-PCR results for the corresponding genes and differences between tissues with each method were concordant. Our results provide the first quantitative atlas of ABC transporters across normal dog tissues and provide a framework for improved understanding of pharmacokinetic and pharmacodynamic variability among dogs receiving anticancer therapies. In addition, this panel will allow evaluation of the potential role that each of these transporters plays in resistance to chemotherapy in vitro and in vivo in dogs, a relevant preclinical model of naturally occurring cancers. Citation Format: Luke A. Wittenburg, Holly Conger, Dominique Ramirez, Daniel Gustafson. Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3871.

An integrated proteomic and peptidomic assessment of the normal human urinome.

Urine represents an ideal source of clinically relevant biomarkers as it contains a large number of proteins and low molecular weight peptides. The comprehensive characterization of the normal urinary proteome and peptidome can serve as a reference for future biomarker discovery. Proteomic and peptidomic analysis of urine can also provide insight into normal physiology and disease pathology, especially for urogenital diseases. We developed an integrated proteomic and peptidomic analytical protocol in normal urine. We employed ultrafiltration to separate protein and peptide fractions, which were analyzed separately using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on the Q-Exactive mass spectrometer. By analyzing six urines from healthy individuals with advanced age, we identified 1754 proteins by proteomic analysis and 4543 endogenous peptides, arising from 566 proteins by peptidomic analysis. Overall, we identified 2091 non-redundant proteins by this integrated approach. In silico protease activity analysis indicated that metalloproteases are predominantly involved in the generation of the endogenous peptide signature. In addition, a number of proteins that were detected in normal urine have previously been implicated in various urological malignancies, including bladder cancer and renal cell carcinoma (RCC). We utilized a highly sensitive proteomics approach that enabled us to identify one of the largest sets of protein identifications documented in normal human urine. The raw proteomics and peptidomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003595.

Shotgun Proteomics of Tomato Fruits: Evaluation, Optimization and Validation of Sample Preparation Methods and Mass Spectrometric Parameters.

An optimized protocol was developed for shotgun proteomics of tomato fruit, which is a recalcitrant tissue due to a high percentage of sugars and secondary metabolites. A number of protein extraction and fractionation techniques were examined for optimal protein extraction from tomato fruits followed by peptide separation on nanoLCMS. Of all evaluated extraction agents, buffer saturated phenol was the most efficient. In-gel digestion [SDS-PAGE followed by separation on LCMS (GeLCMS)] of phenol-extracted sample yielded a maximal number of proteins. For in-solution digested samples, fractionation by strong anion exchange chromatography (SAX) also gave similar high proteome coverage. For shotgun proteomic profiling, optimization of mass spectrometry parameters such as automatic gain control targets (5E+05 for MS, 1E+04 for MS/MS); ion injection times (500 ms for MS, 100 ms for MS/MS); resolution of 30,000; signal threshold of 500; top N-value of 20 and fragmentation by collision-induced dissociation yielded the highest number of proteins. Validation of the above protocol in two tomato cultivars demonstrated its reproducibility, consistency, and robustness with a CV of < 10%. The protocol facilitated the detection of five-fold higher number of proteins compared to published reports in tomato fruits. The protocol outlined would be useful for high-throughput proteome analysis from tomato fruits and can be applied to other recalcitrant tissues.

“Self-cleaving” 2A peptide from porcine teschovirus-1 mediates cleavage of dual fluorescent proteins in transgenic Eimeria tenella

The “self-cleaving” 2A sequence of picornavirus, which mediates ribosome-skipping events, enables the generation of two or more separate peptide products from one mRNA containing one or more “self-cleaving” 2A sequences. In this study, we introduced a single 2A sequence of porcine teschovirus-1 (P2A) linked to two fluorescent protein genes, the enhanced yellow fluorescent protein (EYFP) gene and the red fluorescent protein (RFP) gene, in a single cassette into transgenic Eimeria tenella (EtER). As expected, we obtained two separated protein molecules rather than a fused protein, although the two molecules were translated from the same mRNA carrying a single “self-cleaving” 2A sequence. Importantly, RFP led by a secretion signal was secreted into parasitophorous vacuoles, while EYFP localized mainly to the nucleus of EtER. Our results demonstrate that the “self-cleaving” 2A sequence actively mediated cleavage of polyproteins in the apicomplexan parasite E. tenella.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0351-z) contains supplementary material, which is available to authorized users.

Separation of dynorphin peptides by capillary electrochromatography using a polydiallyldimethylammonium chloride gold nanoparticle-modified capillary.

Dynorphin A (Dyn A) is an endogenous opioid peptide found in blood and central nervous system tissue at very low concentrations. Elevated levels of Dyn A due to different disease states, for example neurodegenerative disease, have been linked to toxic nonopioid activity. CE is a powerful technique that can achieve high-efficiency separations of charged analytes. However, CE has limited use for the analysis of basic proteins and peptides, due to their adsorption onto the inner surface of the fused silica at pHs below their pI. This adsorption can lead to a loss of efficiency, irreproducibility of migration times, and peak tailing. To obviate this problem, a polydiallyldimethylammonium chloride-stabilized gold nanoparticle-coated capillary was investigated for the separation of dynorphin metabolites. The positively charged gold nanoparticles (GNP) minimized unwanted adsorption of the positively charged peptides onto the surface of the fused-silica capillary. Separation efficiency and resolution for opioid peptides Dyn A (1-6), Dyn A (1-7), Dyn A (1-8), Dyn A (1-11), and leu-enkephalin on the GNP-coated capillary column were evaluated under different experimental parameters. The best separation of Dyn A (1-17) and its fragments was achieved using a BGE that consists of 40 mM sodium acetate buffer (pH 5) containing 5% GNP, a field strength of -306 V/cm, and a 75 μm id capillary. The developed method was applied to the separation of tryptic peptide fragments of dynorphin A (1-17).