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Publication date: May 2014 Source:Peptides, Volume 55
Author(s): A.I.R. Nascimento , H.S. Ferreira , D.R. Cerqueira , J.B. Fregoneze
Various studies have investigated the role of central opioid peptides in feeding behavior; however, only a few have addressed the participation of opioids in the control of salt appetite. The present study investigated the effect of intracerebroventricular injections of the δ-opioid antagonist, naltrindole (5, 10 and 20nmol/rat) and the agonist, deltorphin II (2.5, 5, 10 and 20nmol/rat) on salt intake. Two protocols for inducing salt intake were used: sodium-depletion and the central injection of angiotensin II. In addition, the effect of a central δ-opioid receptor blockade on locomotor activity, on palatable solution intake (0.1% saccharin) and on blood pressure was also studied. The blockade of central δ-opioid receptors inhibits salt intake in sodium-depleted rats, while the pharmacological stimulation of these receptors increases salt intake in sodium-replete animals. Furthermore, the blockade of central δ-opioid receptors inhibits salt intake induced by central angiotensinergic stimulation. These data suggest that during sodium-depletion activation of the δ-opioid receptors regulates salt appetite to correct the sodium imbalance and it is possible that an interaction between opioidergic and angiotensinergic brain system participates in this control. Under normonatremic conditions, δ-opioid receptors may be necessary to modulate sodium intake, a response that could be mediated by angiotensin II. The decrease in salt intake following central δ-opioid receptors blockade does not appear to be due to a general inhibition of locomotor activity, changes in palatability or in blood pressure.

NEW YORK (GenomeWeb News) – Researchers from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium have completed a study examining the use of multiple-reaction monitoring mass spec for protein quantitation.

Detailed in a paper published last week in Molecular & Cellular Proteomics, the effort looked at assay development steps required for successful implementation of MRM-MS assays as well as advances in hardware and software that have contributed to improvements in such assays.

Using eight different LC-MS setups across 11 different laboratories, the study quantified more than 100 peptides corresponding to a total of 34 proteins, 27 of which have been linked to cancer.

According to the researchers, the effort demonstrated that "with appropriate attention to experimental design, analytical validation, and suitable quality control methods," highly multiplexed MRM-MS assays can "be implemented by multiple laboratories to provide sensitive, specific, reproducible, and quantitative measurements of proteins and peptides of clinical and biological interest in complex biological matrices."

The study consisted of three phases. In the first, lists of MRM-MS transitions were generated and then tested on 14 different triple quadrupole mass specs (from vendors AB Sciex, Thermo Fisher Scientific, Waters, and Agilent) to select transitions for each peptide best suited to each specific platform. Ultimately, the MRM assays used the three most abundant and interference-free transitions for each peptide measured.

In the second phase of the study, the researchers generated response curves in depleted human plasma. The plasma was digested using Lys-C and trypsin, then spiked with 125 synthetic isotopically labeled peptides, and then used to generate nine-point calibration curves. This phase allowed the researchers to determine variability associated with different instruments.

In the third phase, the researchers generated another set of nine-point response curves by spiking 27 unlabeled target proteins and six unlabeled, previously characterized proteins into depleted, undigested plasma. This allowed them to assess variation due to sample preparation and the digestion process.

The researchers also used isotopically labeled proteins as internal standards in phase II and III to help account for peptide recovery losses due to incomplete digestion and the desalting process. As has been observed by other groups, use of full-length labeled proteins significantly improved assay accuracy and reproducibility, the CPTAC authors noted.

Ultimately, the researchers observed in phase II intralaboratory CVs ranging from 13 percent to 39 percent with a median of 15 percent across the 13 sites performing the assays. Interlaboratory CVs were 31 percent when using the full-length labeled protein standards. These levels of variability represent an improvement over previous CPTAC efforts, the authors noted, citing more rigorous standard operating procedures, use of pre-packed columns and column heaters, and careful monitoring of instrument performance as keys to this improvement.

As expected, the phase III assays, which took into account variability due to sample prep and digestion as well as instrumentation, had higher CVs than in phase II – 58 percent compared to 15 percent.

The researchers also achieved significant improvements in assay sensitivity compared to previous CPTAC work, reaching limits of quantitation at the peptide level of between three- and five-fold better than in earlier studies while increasing the assay multiplex by more than 10-fold. At the protein level, the improvement was more than 20-fold for the seven proteins measured in both studies.

While improvements in mass spec and LC technology contributed to this higher sensitivity, the authors said that they were not as significant contributors as they'd initially expected. Rather, the primary difference was use of immunoaffinity depletion to decrease the plasma sample complexity.

"While use of the newest technology has the potential to improve sensitivity," they wrote, "signal to background biological noise remained the principal limitation on assay sensitivity."

"The two methods that have been clearly demonstrated to decrease biological noise while retaining high analyte signal are fraction MRM [depletion followed by fractionation] and SISCAPA," they added.

Publication date: Available online 23 August 2014 Source:Peptides

Author(s): Jing Tang , Yaqun Fang , Yajun Han , Xuewei Bai , Xiuwen Yan , Yun Zhang , Ren Lai , Zhiye Zhang

Ticks are obligatory blood feeding ectoparasites, which continuously attach to their hosts for 1-2 weeks. There are many biologically active compounds in tick salivary glands interfering host haemostatic system and to successfully obtain blood meal. Several platelet aggregation inhibitors have been identified from ticks. A family of conserved peptides, which were identified from transcriptome analysis of many tick salivary glands, were found to contain unique primary structure including predicted mature peptides of 39-47 amino acid residues in length and a Pro/Glu(P/E)-Pro/His(P/H)- Lys-Gly-Asp(RGD) domain. Given their unique structure and RGD domain, they are considered a novel family of disintegrins that inhibit platelet aggregation. One of them (YY-39) was tested for its effects on platelets and thrombosis in vivo. YY-39 was found effectively to inhibit platelet aggregation induced by adenosine diphosphate (ADP), thrombin and thromboxane A2 (TXA2). Furthermore, YY-39 blocked platelet adhesion to soluble collagen and bound to purified GPIIb/IIIa in a dose-dependent manner. In in vivo experiments, YY-39 reduced thrombus weight effectively in a rat arteriovenous shunt model and inhibited thrombosis in a carrageenan-induced mouse tail thrombosis model. Combined with their prevalence in ticks and platelet inhibitory functions, this family of peptides might be conserved tick anti-haemostatic molecules.

By Waleed Danho, PhD, Director, Boulder Peptide Society

Protein therapeutics is gaining headway in the treatment and prevention of a variety of maladies. Critical to the success and growing use of biologic-level pharmaceuticals is the development of methods for their precise synthesis. There are emerging technologies for the total synthesis of proteins, with a special focus Read more...

Publication date:

Rolling in the MUC1

Writing in the journal Vaccines last year, Behjatolah Monzavi-Karbassi, Ph.D., and colleagues reviewed some of the challenges associated with development of glycoprotein-based vaccines. Citing the favorite target of glycan-based vaccine developers, MUC1, cancer vaccines targeting this antigen had been based on carrier-conjugated unglycosylated MUC1 tandem repeat peptides or carrier-conjugated glycosylated epitopes. These efforts proved largely unsuccessful. MUC1 is a high-molecular-weight glycoprotein having few isoforms encoding both a transmembrane and cleavage-truncated secreted (soluble) product. Both forms are expressed by more than 90% of solid epithelial tumor cancers as well as most common nonsolid tumors including multiple myeloma. Read more...

An update for human blood plasma pretreatment for optimized recovery of low-molecular-mass peptides prior to CE–MS and SPE–CE–MS

Journal of Separation Science, 2013, 36, 3896-3902
Laura Pont, Fernando Benavente, José Barbosa, Victoria Sanz-Nebot

Abstract: Protein precipitation and centrifugal filtration are well-established methods for concentrating and purifying peptides with a low relative molecular mass (M_r) from human blood plasma before proteomic and peptidomic studies using high-performance separation techniques, but there is little information on peptide recoveries. Read more...