The low molecular weight (LMW; <10kDa)* plasma peptidome has been considered a source of useful diagnostic biomarkers and potentially therapeutic molecules, as it contains many cytokines, peptide hormones, endogenous peptide products and potentially bioactive fragments derived from the parent proteome. The small size of the peptides allows them almost unrestricted vascular and interstitial access, and hence distribution across blood-brain barriers, tumour and other vascular permeability barriers. Therefore, the peptidome may carry specific signatures or fingerprints of an individual's health, wellbeing or disease status. This occurs primarily because of the advantage the peptidome has in being readily accessible in human blood and/or other biofluids. However, the co-expression of highly abundant proteins (>10kDa) and other factors present inherently in human plasma make direct analysis of the blood peptidome one of the most challenging tasks faced in contemporary analytical biochemistry. A comprehensive compendium of extraction and fractionation tools has been collected concerning the isolation and micromanipulation of peptides. However, the search for a reliable, accurate and reproducible single or combinatorial separation process for capturing and analysing the plasma peptidome remains a challenge. This review outlines current techniques used for the separation and detection of plasma peptides and suggests potential avenues for future investigation. This article is part of a Special Issue entitled: HUPO 2014.

OBJECTIVE: To determine the usefulness of brain-derived neurotrophic factor (BDNF) as a diagnostic biomarker for colorectal cancer (CRC).MATERIALS AND METHODS: ELISA immunoassay was used to examine BDNF concentrations in the sera of two different retrospective cohorts consisting of CRC patients and age/gender matched controls. Cohort 1 consisted of 99 controls and 97 CRC patients, whereas cohort 2 consisted of 47 controls and 91 CRC patients.RESULTS: In cohort 1, the median concentration of BDNF was significantly (p < 0.0001) lower in CRC patient samples (18.8 ng/mL, range 4.0-56.5 ng/mL) than control samples (23.4 ng/mL, range 3.0-43.1 ng/mL). This finding was validated in an independent patient cohort (CRC patients: 23.0 ng/mL, range 6.0-45.9 ng/mL; control patients: 32.3 ng/mL, range 14.2-62.4 ng/mL). BDNF concentrations did not differ significantly between Dukes' staging in the patient cohort, however patients with Stages A, B, C and D (p < 0.01 for each stage) tumours had significantly reduced BDNF levels compared to healthy controls. Receiver operating characteristic analysis was performed to determine the ability of BDNF to discriminate between healthy controls and those with CRC. At 95% specificity, BDNF concentrations distinguished CRC patients with 25% and 18% sensitivity, respectively, in cohorts 1 and 2 (cohort 1: AUC = 0.79, 95% CI 0.70-0.87; cohort 2: AUC = 0.69, 95% CI 0.61-0.76).CONCLUSION: The serum levels of BDNF were significantly lower in colorectal cancer patients when compared to a control population, and this did not differ between different Dukes' stages.

Molecular dynamics (MD) simulations and far-infrared (far-IR) spectroscopy were combined to study peptide binding by the second PDZ domain (PDZ1) of MAGI1, which has been identified as an important target for the Human Papilloma Virus. PDZ1 recognizes and binds to the C-terminal end of the E6 protein from high-risk Human Papilloma Virus. The far-IR spectra of two forms of the protein, an unbound APO form and a HOLO form (where the PDZ1 is bound to an 11-residue peptide derived from the C terminus of HPV16 E6), were obtained. MD simulations were used to determine the most representative structure of each form and these were used to compute their respective IR spectra by normal mode analysis. Far-UV circular dichroism spectroscopy was used to confirm the secondary structure content and the stability through temperature-dependent studies. Both the experimental and calculated far-IR spectra showed a red shift of the low-frequency peaks upon peptide binding. The calculations show that this is coincident with an increased number of hydrogen bonds formed as the peptide augments the protein β-sheet. We further identified the contribution of surface-bound water molecules to bands in the far-IR and, through the calculations, identified potential pathways for allosteric communication. Together, these results demonstrate the utility of combining far-IR experiments and MD studies to study peptide binding by proteins.

B15 The matrix metalloproteinases (MMP) and insulin-like growth factors (IGF) are known to be involved in the process of tumor invasion and progression (1, 2). Previous research has shown that the concentration of these proteins can change in the sera of cancer patients (3, 4). Our objective was to investigate the potential diagnostic value of circulating MMPs and IGFs in subjects with colorectal cancer. Enzyme linked immunoassays were used to quantify the concentrations of MMP-2, -7, -9, IGF-I, -II and IGF-BP3 in the plasma and serum of 51 preoperative colorectal cancer patients and 50 control subjects. Colorectal cancer subjects had lower serum MMP-2 (p

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST-beta-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST-immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC-MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads.

Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

The Drosophila SINA (seven in absentia) protein and its mammalian orthologs (Siah, seven in absentia homolog) are RING domain proteins that function in E3 ubiquitin ligase complexes and facilitate ubiquitination and degradation of a wide range of cellular proteins, including beta-catenin. Despite these diverse targets, the means by which SINASiah recognize substrates or binding proteins has remained unknown. Here we identify a peptide motif (RPVAxVxPxxR) that mediates the interaction of Siah protein with a range of protein partners. Sequence alignment and mutagenesis scanning revealed residues that are important to this interaction. This consensus sequence correctly predicted a high-affinity interaction with a peptide from the cytoskeletal protein plectin-1 (residues 95-117). The unusually high-affinity binding obtained with a 23-residue peptide (K(Dapp) = 29 nM with SINA) suggests that it may serve as a useful dominant negative reagent for SINASiah proteins.

BioEssaysVolume 24, Issue 11 p. 1080-1081 ErratumFree Access Nice E. Biosensors: book review of chemical sensors and biosensors, by Brian R. Eggins. bioessays 2002; 24:977–978 Edouard C. Nice, Corresponding Author Edouard C. Nice Protein Biosensing Laboratory, Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch Melbourne, Victoria, 3050, AustraliaProtein Biosensing Laboratory, Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch Melbourne, Victoria, 3050, Australia.Search for more papers by this author Edouard C. Nice, Corresponding Author Edouard C. Nice Protein Biosensing Laboratory, Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch Melbourne, Victoria, 3050, AustraliaProtein Biosensing Laboratory, Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch Melbourne, Victoria, 3050, Australia.Search for more papers by this author First published: 17 October 2002 https://doi.org/10.1002/bies.10197AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume24, Issue11November 2002Pages 1080-1081 RelatedInformation

The selective introduction of functional groups on the surface of silicon nitride/silicon oxide nanostructures was studied. Chemical strategies based on organosilane, Si–H and N–H reactivities were assayed. Among these strategies, the use of glutaraldehyde to selectively immobilize biomolecules only on the silicon nitride part of the chip surface was the most effective for the covalent attachment of proteins, maintaining also their bioavailability. The biomolecule surface coverage results up to 80% and the modification is selective versus silicon oxide; the biomolecule attaching only to silicon nitride and leaving the silicon oxide area of the device unmodified. The effectiveness of our novel selective surface modification procedure is also supported by comparing experimental and numerical calculations of the optical performance of a label-free optical ring resonator based on Si3N4/SiO2 slot-waveguides.

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF) and human transforming growth factor-α (h-TGF-α) with high affinity despite significant differences in the amino acid sequences of the ligands and the receptors [1]. In contrast, the chicken EGFR can discriminate between mEGF or hEGF and hTGF-α, the EGFs having approximately 100-fold lower affinity [2]. The regions responsible for this poor binding are known to be Arg45 in hEGF [3] and the L2 domain in the chicken EGFR [4]. In this study we have produced a truncated form of the hEGFR ectodomain comprising domains L1/CR1/L2 plus the first module of the second Cys-rich region, CR2 (residues 1–501, sEGFR501). This truncated receptor was used to characterise its ligand-binding properties, including its ability to form ligand-induced dimers and to act as an inhibitor of EGF-stimulated mitogenic responses in BaF cells transfected with the EGFR [5]. In addition, based on a model of the EGFR extracellular domain [6] we have generated three mutants of sEGFR501 with single position substitutions at Glu367, Gly441 or Glu472 to Lys, the residue found in the corresponding positions in the chicken EGFR, to investigate the residues responsible for ligand discrimination.

Large numbers of colon tumors stem from mutations in the gene coding for the production of the adenomatous polyposis coli (APC) tumor suppressor protein. This protein contains a coiled-coil N-terminal domain that is known to be responsible for homodimerization. Previous work by others has led to the design of a specific 54-residue anti-APC peptide (anti-APCp(1)) that dimerizes preferentially with this domain. We have undertaken the chemical synthesis of a modified form of this peptide (anti-APCp(2)) that bears a biotin moiety at its N-terminus for use in subsequent ligand-binding analysis studies. The peptide was subjected to comprehensive chemical characterization to confirm its purity. Secondary structural analysis by circular dichroism spectroscopy and Fourier transform infrared spectroscopy indicated that the peptide could assume a wide range of potential conformations, depending upon the precise microenvironment. Significantly, a stable chi -helical structure was generated when the solvent conditions supported intramolecular salt-bridge formation along the helix barrel. The biotinylated anti-APCp(2) was immobilized onto a streptavidin sensor surface, in a specific orientation leaving all amino acids available to form a coiled structure. In one experiment, injection of colonic cell lysate extracts (LlM1215) onto a size-exclusion column resulted in the isolation of a high molecular mass protein peak (> 600 kDa) that reacted specifically with the. immobilized anti-APCp(2) on the biosensor surface. In another experiment, a high molecular mass protein (M-r> 250 kDa on SDS-PAGE) could be specifically immunoprecipitated from this peak using either the anti-APCp(2) peptide or an anti-APC polyclonal antibody. This demonstrates the specific interaction between the anti-APCp(2) peptide and native APC and highlights the potential use of the former peptide in a multidimensional micropreparative chromatographic/biosensor/proteomic protocol for the purification of APC alone and APC complexed with different biopolymers in various cell lines, and stages of tumor development.

Drugs that inhibit important protein-protein interactions are hard to find either by screening or rational design, at least so far. Most drugs on the market that target proteins today are therefore aimed at well-defined binding pockets in proteins. While computer-aided design is widely used to facilitate the drug discovery process for binding pockets, its application to the design of inhibitors that target the protein surface initially seems to be limited because of the increased complexity of the task. Previously, we had started to develop a computational combinatorial design approach based on the well-known 'multiple copy simultaneous search' (MCSS) procedure to tackle this problem. In order to identify sequence patterns of potential inhibitor peptides, a three-step procedure is employed: first, using MCSS, the locations of specific functional groups on the protein surface are identified; second, after constructing the peptide main chain based on the location of favorite locations of N-methylacetamide groups, functional groups corresponding to amino acid side chains are selected and connected to the main chain C(alpha) atoms; finally, the peptides generated in the second step are aligned and probabilities of amino acids at each position are calculated from the alignment scheme. Sequence patterns of potential inhibitors are determined based on the propensities of amino acids at each C(alpha) position. Here we report the optimization of inhibitor peptides using the sequence patterns determined by our method. Several short peptides derived from our prediction inhibit the Ras--Raf association in vitro in ELISA competition assays, radioassays and biosensor-based assays, demonstrating the feasibility of our approach. Consequently, our method provides an important step towards the development of novel anti-Ras agents and the structure-based design of inhibitors of protein--protein interactions.

Although it is 75 years since relaxin was first described [1], and almost 20 years since the cDNA sequences encoding for rat, porcine and human relaxin were reported [2,3,4], the isolation and characterisation of the cognate receptor/s, despite extensive investigation [5,6], has to date defied elucidation. Since the receptor would appear to be the key to a fuller understanding of the molecular mechanisms of action of relaxin [6], which is fundamental for structure-based design of agonists and antagonists with therapeutic potential, new approaches for its isolation warrant investigation.

Mutational analysis of the cRaf-1 Ras binding domain (RBD) identified several point mutants with elevated Ras binding. Detailed examination of the binding kinetics of one mutant (A85K) suggests that it associates with a greater range of isomeric conformers of v-H-Ras than wt-RBD. At limiting v-H-Ras concentrations, saturation binding to A85K-RBD is higher than to wt-RBD. Notably, in assay systems where the RBD concentration is limiting, no difference exists between wt-RBD and A85K-RBD saturation levels in the presence of a sufficiently large molar excess of Ras. The inability of wt-RBD to saturate all bindable Ras/GTP (defined by its binding to A85K-RBD) suggests that Ras/GTP exists as several isoforms and that only a minority of these isoforms are capable of associating with wt-RBD. These findings provide the first experimental evidence in support of functionally distinct Ras/GTP isoforms. We also describe a novel analysis of such isoforms.

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2, In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions, Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2, One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction, Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF, In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members; These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.