A strategy for preparing high relaxivity, metabolically stable peptide-based MR contrast agents is described.The chemical and topological diversity of peptides offer tremendous possibilities to identify new diagnostic imaging compounds. Peptides have been widely used to target an imaging probe to a specific protein or receptor and thereby provide greater specificity. Typically an imaging reporting moiety (e.g. positron emitter, gamma emitter, paramagnetic ion, near infrared fluorophore) is conjugated to the peptide. The site of conjugation, the linker, and the type of imaging reporter all play a role in determining biological activity and pharmacokinetics. 1, 2 For peptide-based magnetic resonance imaging (MRI) contrast agents, an additional factor is detection sensitivity of the imaging probe. 3 Multiple copies of the MR active reporter, typically a gadolinium complex, are required to provide robust image contrast.An additional major challenge to creating new drugs from peptides is peptide degradation by endogenous peptidases. There are numerous medicinal chemistry approaches to improve peptide stability, biological activity, and/or bioavailability that increase in complexity from modified peptides to pseudopeptides to small molecule peptidomimetics. 4,5 In this report, we explore the potential of using the imaging reporter to block peptidase activity.We, 6-9 and others, 10-14 have been interested in developing gadolinium-based peptide-targeted MR imaging agents. Compared to other modalities, MRI provides a favorable combination of high spatial resolution, depth penetration, and lack of ionizing radiation. Unlike nanoparticles, these relatively small molecules can rapidly reach targets in extravascular spaces and can be readily excreted through the kidneys to reduce/avoid long-term gadolinium retention and toxicity. On the other hand, extravasation into the kidneys and liver exposes these compounds to a range of peptidases.There is some flexibility as to where and how the gadolinium chelates are conjugated to the peptide. Conjugation is possible at the N-or C-terminus and/or within the peptide structure. 6 We recently reported some fibrin-specific peptides conjugated with one or four [Gd (DTPA)] 2− moieties. 8 The construct with highest affinity had two peptides linked via their Nterminus to a GdDTPA tetramer, i.e. Pep N -Gd 4 -N Pep, termed EP-1084 (Cmpd 1 in Scheme

An efficient method for data processing and interpretation is needed to support and extend disulfide mass-mapping methodology based on partial reduction and cyanylation-induced cleavage to proteins containing more than four cystines. Here, the concept of "negative signature mass" is introduced as the novel feature of an algorithm designed to identify the disulfide structure of a cystinyl protein given an input of mass spectral data and an amino acid sequence. The "negative signature mass" process is different from the conventional approach in that it does not directly rule-in disulfide linkages, but rather eliminates linkages from a list of all possible theoretical linkages, with the goal of ruling out enough linkages so that only one disulfide structure can be constructed. The operating principles and the effectiveness of the algorithm are described in the context of analyzing ribonuclease A, a 124-residue protein containing eight cysteines in the form of four cystines (disulfides).

Mass mapping analysis based on cyanylation and CNinduced cleavage indicates that the two cysteine residues in the C-terminal extension of the B subunit of the light-activated pea leaf chloroplast glyceraldehyde-3-phosphate dehydrogenase form a disulfide bond. No evidence was found for a disulfide bond in the A subunit, nor was there any indication of a second disulfide bond in the B subunit. The availability of the structure of the extended glyceraldehyde-3-phosphate dehydrogenase from the archaeon Sulfolobus solfataricus allows modeling of the B subunit. As modeled, the two cysteine residues in the extension are positioned to form an interdomain disulfide cross-link.The NADP-linked chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) 1 (EC 1.2.1.13) of flowering plants consist of two subunits. The A subunit is similar in length and in sequence to the eucaryotic and eubacterial NAD-linked enzyme (EC 1.2.1.12) protomers. The B subunit has a 27-to 29-residue C-terminal extension that contains two invariant cysteines (Fig. 1). B subunits have only been found in angiosperms. In species ranging from Cyanobacteria to angiosperms, NADP-linked glyceraldehyde-3-phosphate dehydrogenases are light-activated (1, 2). Activation involves the reduction of an inhibiting disulfide bond or bonds. Although modeling indicates that two Cys residues in the A subunit are almost certainly responsible for the redox sensitivity of the enzyme in green algae and nonflowering plants (3), the presence of the conserved Cys pair in the extended angiosperm B subunit suggests that it too might be involved in redox regulation. The purpose of the present experiments was to determine which cysteine residues in the pea chloroplast glyceraldehyde-3-phosphate dehydrogenase form cystines and are responsible for the redox sensitivity of the enzyme. MATERIALS AND METHODSEnzyme Isolation-The wild type NADP-linked glyceraldehyde-3-phosphate dehydrogenase used in the mass mapping experiments was purified from pea plants by a modification of the method of Anderson et al. (4). Prior to the acetone fractionation step, the suspended MgCl 2 -polyethylene glycol fraction was passed through a DEAE column (10 ml of Sigma Fast Flow DEAE) in 10 mM KHPO 4 , pH 7.8 buffer. The glyceraldehyde-3-phosphate dehydrogenase in this fraction was not retained on the column. It emerged in a milky excluded fraction. After acetone fractionation, the cloudy 50% acetone supernatant was applied to a second 10-ml DEAE column in 10 mM Tris-HCl, pH 7.8, and the column was washed overnight with the Tris buffer and eluted with a linear phosphate gradient (200 ml of 10 mM KHPO 4 , pH 7.8 and 200 ml of 0.4 M KHPO 4 , pH 7.0). In some experiments, including those represented by Figs. 2 and 3, 10 mM mercaptoethanol was included in the buffers. In other experiments, including the experiments represented by Figs. 4 and 5, mercaptoethanol was omitted. Essentially identical results were obtained with or without inclusion of mercaptoethanol in the buffers during isolation. Chromatograph...

Our cyanylation (CN)-based methodology for determining disulfide structure of cystinyl proteins overcomes the limitations of conventional proteolytic methods. However, the CN-based method has the potential drawback that occasionally some CN-induced cleavage fragments may not be detected. We show that CN-based methods can overcome the failure to detect fragments by demonstrating the existence of small 'signature sets' of fragments. The link between signature sets and the robustness of CN-based methodology is validated by two case studies.

RNA-binding proteins (RBPs) play important roles in regulating gene expression and dysregulation of RBPs have been observed in various types of cancer. However, the role of RBPs during glioma progression, and particular in Chinese patients, is only starting to be unveiled. Here, we systematically analyzed the somatic mutation, gene expression patterns of 2949 RBPs during glioma progression. Our comprehensive study reveals several of highly mutated genes (such as ATRX, TTN and SETD2) and differentially expressed genes (such as KIF4A, TTK and CEP55). Integration of the expression of RBPs and genes, we constructed a regulatory network in glioma and revealed the functional links between RBPs and cancer-related genes. Moreover, we identified the prognosis spectrum of RBPs during glioma progression. The expression of a number of RBPs, such as SNRPN and IGF2BP3, are significantly associated with overall survival of patients in all grades. Taken together, our analyses provided a valuable RBP resource during glioma progression, and revealed several candidates that potentially contribute to development of therapeutic targets for glioma.