Tri- and dipeptides identification in whey protein and porcine liver protein hydrolysates by fast LC-MS/MS neutral loss screening and de novo sequencing.

We describe a fast (5 min) liquid chromatography tandem mass spectrometry method (LC-MS/MS) based on a 46 Da neutral loss of formic acid (H2 O and CO) to identify tri- and dipeptides (DIPEP) in whey protein and porcine liver protein hydrolysates and confirmed by further de novo sequencing. Sample solutions were acidified to favor [dipep + H]+ ions, and a m/z range of 50-300 was used to improve sensitivity. All dipeptide candidates were selected based on all possibilities of the 20 amino acid combinations, and their collision-induced dissociation fragments were screened via de novo sequencing. To determine their biological activities, sequenced dipeptides were compared with the Biopep database and other data from literature. Altogether, 18 dipeptides and 7 tripeptides were identified from the whey protein hydrolysate; they seemed to be broadly active, and peptides were identified as active dipeptidyl peptidase IV inhibitors and active angiotensin-converting enzyme (ACE), according to available information. Porcine liver hydrolysate showed 14 dipeptides which exhibit similar biological activities to whey protein hydrolysate.

Toxic Peptides in Populations of Two Pergid Sawflies, Potential Biocontrol Agents of Brazilian Peppertree.

Determination of the safety of agents prior to release is one of the most important research goals in biological control. In addition to concerns for the safety of non-target plants, determination of the potential toxic properties of new agents needs to be assessed. Numerous phytophagous insects are defended by chemicals against the attack of natural enemies. Some of these defensive compounds could pose an environmental risk if an agent is released. Here, larval populations of two pergid sawflies, Heteroperreyia hubrichi and H. jorgenseni, were analyzed by LC-MS/MS to investigate whether they contain alleged toxic peptides. The first species is a potential candidate for biological control of the invasive weed Brazilian peppertree in Florida and Hawaii. The chemical analyses revealed the presence of the peptides pergidin (Perg), 4-valinepergidin (VPerg), dephosphorylated pergidin (dpPerg), lophyrotomin (LGln and LGlu). The effect of sawfly population for each species was significantly influencing peptide concentration. All peptides occurred at lower concentrations compared with purportedly toxic species of this sawfly family. However, the concentrations of the peptides are of concern for the welfare of wildlife and livestock that would be exposed to these species. These results demonstrate that release of this biological control agent in the invaded range may pose an environmental threat.

Proteomic and peptidomic profiling of Brazilian artisanal 'Coalho' cheese.

BACKGROUND: Artisanal 'Coalho' cheese is a product typically popular in the Brazilian north-eastern region. Production of this cheese represents about 9.2% of the internal crude product of Pernambuco State. Several peptides are generated from hydrolysis of αS1 -, αS2 -, ß-, and κ-caseins during manufacture of this cheese. The commercial importance of Brazilian artisanal 'Coalho' cheese justifies the examination of both the protein and peptide profiles of cheeses from six cities of the semi-arid region of Pernambuco State, Brazil. RESULTS: SDS-PAGE of the aqueous extracts of 'Coalho' cheeses (WSP) showed bands of lactoferrin, ß-lactoglobulin, ß-lactoglobulin (dimer), α-lactoalbumin, bovine serum albumin, α-casein, ß-casein, κ-casein and para-κ-casein. A total of 57 to 72 peptides were confirmed by mass spectra in the different samples of 'Coalho' cheese which 32 known peptides (11 from αS1 -casein, three from αS2 -casein, 15 from ß-casein and three from κ-casein), comprising seven caseinphosphopeptides. Among the unidentified peptides, three showed high intensity peaks in all 'Coalho' cheeses studied (with molecular weights of 1597, 1725/1726, 2778/2779 Da). CONCLUSION: The proteomic studies revealed peptides that may represent molecular markers or fingerprints for investigating the quality control and regional characterisation of these 'Coalho' cheeses. © 2016 Society of Chemical Industry.

In the picture: disulfide-poor conopeptides, a class of pharmacologically interesting compounds

During evolution, nature has embraced different strategies for species to survive. One strategy, applied by predators as diverse as snakes, scorpions, sea anemones and cone snails, is using venom to immobilize or kill a prey. This venom offers a unique and extensive source of chemical diversity as it is driven by the evolutionary pressure to improve prey capture and/or to protect their species. Cone snail venom is an example of the remarkable diversity in pharmacologically active small peptides that venoms can consist of. These venom peptides, called conopeptides, are classified into two main groups based on the number of cysteine residues, namely disulfide-rich and disulfide-poor conopeptides. Since disulfide-poor conotoxins are minor components of this venom cocktail, the number of identified peptides and the characterization of these peptides is far outclassed by its cysteine-rich equivalents. This review provides an overview of 12 families of disulfide-poor peptides identified to date as well as the state of affairs.(AU)

In the picture: disulfide-poor conopeptides, a class of pharmacologically interesting compounds

During evolution, nature has embraced different strategies for species to survive. One strategy, applied by predators as diverse as snakes, scorpions, sea anemones and cone snails, is using venom to immobilize or kill a prey. This venom offers a unique and extensive source of chemical diversity as it is driven by the evolutionary pressure to improve prey capture and/or to protect their species. Cone snail venom is an example of the remarkable diversity in pharmacologically active small peptides that venoms can consist of. These venom peptides, called conopeptides, are classified into two main groups based on the number of cysteine residues, namely disulfide-rich and disulfide-poor conopeptides. Since disulfide-poor conotoxins are minor components of this venom cocktail, the number of identified peptides and the characterization of these peptides is far outclassed by its cysteine-rich equivalents. This review provides an overview of 12 families of disulfide-poor peptides identified to date as well as the state of affairs.

Co-occurrence of microcystin and microginin congeners in Brazilian strains of Microcystis sp.

Species of Microcystis are the most common bloom-forming cyanobacteria in several countries. Despite extensive studies regarding the production of bioactive cyanopeptides in this genus, there are limited data on isolated strains from Brazil. Three Microcystis sp. strains were isolated from the Salto Grande Reservoir (LTPNA01, 08 and 09) and investigated for the presence of mcy genes, microcystins and other cyanopeptides. Microcystin and microginin production was confirmed in two isolates using high-resolution tandem mass spectrometry after electrospray ionization (ESI-Q-TOF), and the structures of two new microginin congeners were proposed (MG756 Ahda-Val-Leu-Hty-Tyr and MG770 MeAhda-Val-Leu-Hty-Tyr). The biosynthesis profile of the identified cyanopeptides was evaluated at different growth phases via a newly developed HPLC-UV method. Results demonstrated no substantial differences in the production of microcystins and microginins after data normalization to cell quota, suggesting a constitutive biosynthesis. This study represents the first confirmed co-production of microginins and microcystins in Brazilian strains of Microcystis sp. and highlights the potential of Brazilian cyanobacteria as a source of natural compounds with pharmaceutical interest.

Development and validation of a bioanalytical LC-MS method for the quantification of GHRP-6 in human plasma.

Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH2, MW=872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with ¹³C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC-MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R² higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotesvasodilatation mediated by both bradykinin B2 and M1 muscarinicacetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venomglands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme(ACE) described. Bj-PRO-5a (

Mechanisms of the beneficial effect of hypertonic saline solution in acute pancreatitis.

Administration of hypertonic saline (HS) solution to rats with acute pancreatitis (AP) decreases mortality and systemic inflammation. We hypothesized that these effects are related not only to systemic inflammatory reduction, but also to a reduction of the pancreatic lesion. Acute pancreatitis was induced in Wistar rats by injection of 2.5% sodium taurocholate. Animals were divided in groups: without AP, not treated AP, AP treated with NaCl 0.9%, and AP treated with NaCl 7.5%. Trypsinogen activation peptides and amylase activity were increased in ascitic fluid and serum and were not affected by treatment with HS. Pancreatic inflammation was evaluated by increased myeloperoxidase activity, malondialdehyde formation, and histopathology for severity of pancreatic lesions. The HS did not affect these parameters. Expression of cyclooxygenase 2 and inducible nitric oxide synthase was markedly increased in the pancreas of the AP group and was reduced by treatment with HS. This treatment also reduced the levels of TNF-α and IL-6 but not of IL-10 in the pancreatic tissue. These results show that HS modulates cytokine production and expression of enzymes responsible for inflammatory mediator production in the pancreas without affecting the severity of the pancreatic lesions.

Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii.

We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.

Identification of three novel peptides isolated from the venom of the neotropical social wasp Polistes major major.

Three novel peptides designated as PMM1, PMM2, and PMM3 were isolated and characterized from the venom of the social wasp Polistes major major, one of the most common wasps in the Dominican Republic. By Edman degradation, and MALDI-TOF and ESI-QTOF mass spectrometry, the primary sequences of these peptides were established as follows: PMM1, H-Lys-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH (1357.77 Da); PMM2, H-Ile-Asn-Trp-Lys-Lys-Ile-Ala-Ser-Ile-Gly-Lys-Glu-Val-Leu-Lys-Ala-Leu-NH2 (1909.19 Da); and PMM3, H-Phe-Leu-Ser-Ala-Leu-Leu-Gly-Met-Leu-Lys-Asn-Leu-NH2 (1317.78 Da). The suggested sequences were confirmed by MS analysis of peptide fragments obtained by enzymatic digestion. The peptide PMM1 is a lysyl-arginyl-Thr(6)-bradykinine that belongs to the wasp kinins group. The sequence of the PMM2 peptide is unique; it resembles somewhat the tetradecapeptide amides of the mastoparan group; however, the chain is extended by three additional amino acid residues. The sequence of PMM3 dodecapeptide is homologous to the peptides of the wasp chemotactic group.

Improved photostable FRET-competent biarsenical-tetracysteine probes based on fluorinated fluoresceins.

We have developed fluoro-substituted versions of the biarsenical-tetracysteine label FlAsH, exhibiting significant improvements in important properties over the original fluorescein derivative. In complexes with tetracysteine targets, F2FlAsH exhibits 50 times improved photostability, lower pH sensitivity, higher absorbance and quantum yield than FlAsH, and F4FlAsH adds a new color to the palette of biarsenical dyes. The two probes also provide a new FRET pair with a larger Ro value (54 A) than any previously obtained with biarsenical dyes.

Thrombin generation in platelet-poor plasma is normal in patients with hereditary mucocutaneous haemorrhages.

Mild hereditary bleeding disorders presenting with mucocutaneous haemorrhages are usually difficult to diagnose. We measured thrombin generation in platelet-poor plasma (TG-PPP) in 206 patients with a clinically unequivocal bleeding tendency: 45 with von Willebrand disease (vWD), 49 with platelet aggregation/secretion defects (PASD), 10 with a combination of both and 102 who did not fit the diagnostic criteria for any known haemostatic disorder. TG-PPP was not significantly different from controls in all patient groups, indicating that an abnormality in the plasmatic clotting system is unlikely to contribute to the bleeding in patients with type 1 vWD and PASD. In patients with undiagnosed mild hereditary bleeding disorders, there must be other mechanisms which explain the abnormal haemorrhagic tendency, most likely as yet unrecognized defects in platelet-vessel wall interaction. As a next step we plan to investigate thrombin generation in PRP.

Influence of pancreatic enzyme content on experimental acute pancreatitis.

INTRODUCTION: Some studies demonstrate the crucial role of proteases in the pathogenesis of acute pancreatitis (AP). Systemic release of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) has been demonstrated in AP, yet the mechanism of activation remains unclear. Furthermore, it is not known if the amount of pancreatic enzyme in the pancreas determines the production of proinflammatory cytokines. AIM: To determine whether there is a link between the pancreatic enzyme content and the production of cytokines and consequently the systemic lesions observed in AP. METHODOLOGY: Forty-seven animals were divided into three groups: group I had a high pancreatic enzyme level (with and without AP), group II had a low pancreatic enzyme level (cerulein infusion: 0.133 microg x kg(-1) x h(-1)) (with and without AP), and group III were the controls. AP was induced by injection of 5% sodium taurocholate into the pancreatic duct. To evaluate the pancreatic enzyme contents before AP, trypsinogen and amylase analysis was carried out on pancreatic tissue collected after the animals were killed. Two hours after induction of AP, concentrations of pancreatic enzymes and trypsinogen activation peptide (TAP) in serum, ascitic fluid, and pancreatic tissue were determined. The ascitic fluid was assayed for TNF-alpha and the serum was assayed for IL-6 with ELISA kits. Systemic lesions were sought on the basis of hepatic mitochondrial respiratory function measured polarographically. RESULTS AND CONCLUSION: The administration of physiological doses of cerulein diminishes the pancreatic enzyme and TAP levels, the production of proinflammatory cytokines, and the liver mitochondrial dysfunction observed in AP, suggesting that the pancreatic enzyme content is an important factor in the severity of AP.

Identification of mimotopes of platelet autoantigens associated with autoimmune thrombocytopenic purpura.

GPIIb/IIIa, the human platelet glycoprotein complex, is the autoantigen most commonly recognized by autoantibodies in autoimmune thrombocytopenic purpura (AITP). Two murine monoclonal antibodies (mAbs), namely Y2/51 and 5B12, directed against gpIIIa and gpIIb/IIIa, respectively, and rabbit anti-human platelet polyclonal antibodies have been used to select AITP-related epitopes from a phage display peptide library expressing random dodecapeptides in the pIII coat protein of M13 phage. The selected phage clones were tested by ELISA for binding to rabbit anti-human platelet antibodies as well as to sera from AITP patients. Seven clones reacted strongly with rabbit anti-human platelet antibodies, and four clones reacted with sera from AITP patients. Some homology between peptide inserts sequences of selected clones and human platelet gpIIIa and gpIb were found.

Sequencing of argentinated peptides by means of electrospray tandem mass spectrometry.

A strategy for semiautomatic sequencing of argentinated (silver-containing) oligopeptides has been developed. Sequencing is based on a search algorithm that identifies a triplet peak relationship in a product ion spectrum of the [M + Ag]+ ion of an oligopeptide. The ions that constitute a triplet are [bn + OH + Ag]+, [bn - H + Ag]+, and [a(n) - H + Ag]+, which are separated by 18 and 28 m/z units, respectively. The difference in the m/z values of adjacent triplets identifies the residue that is "cleaved". Observation of the [yn + H + Ag]+ ion containing the cleaved residue confirms the assignment. Sequencing of argentinated tryptic peptides may prove useful for automated proteome analysis via the sequence tag method.

Immunohistopathological characterizatin of spontaneous metastases in a human lung mucoepidermoid adenocarcinoma (HLMC) xenograft.

The most common clinical form of lung cancer is a disseminated disease with distant metastases; several years of cancer progression precede presentation, and this ultimately limits the efficacy of curative therapy. In this immunohistochemical study, we examined a mucinous adenocarcinoma cell line, maintained by xenogeneic transplantation, and a spontaneous metastatic variant which produces distant tumors (in liver, spleen and kidney). The aim was to investigate possible parameters which characterize the metastatic process. Histopathological comparison between the two subcutaneous transplanted tumor lines showed that both lines presented a similar cellular morphology, a different pattern of cellular growth and an increased vascularization in the metastatic line with respect to its parent. All the tumor sections expressed differential immune reactivity with monoclonal antibodies against Lewis y (MAb C14), sialyl-Lewis x (MAb SNH3) and Lewis x (MAb FH2) determinants. Neither expressed MUC 1 mucins detectable with monoclonal antibodies reactive with the mucin protein core (MAbs C595 and SM3) nor was carcinoembryonic antigen (MAb C365) expressed. Neoplastic cells were reactive with an anti-pan cytokeratin monoclonal antibody confirming their epithelial histogenesis. Our findings have been evaluated with respect to defining metastatic phenotypes in lung cancer by examination of distinct histopathological and immunological parameters.

Confocal microscopy of a newly identified protein associated with heart development in the Mexican axolotl.

Recessive mutant gene c for "'cardiac nonfunction" in the mexican axolotl, Ambystoma mexicanum, results in a failure of affected embryos to develop contracting hearts. Mutant embryos survive approximately 4 weeks after fertilization, but eventually die from a lack of circulation. Morphological studies show that mutant hearts lack organized sarcomeric myofibrils. This abnormality can be corrected by co-culturing early mutant hearts with normal anterior endoderm/mesoderm tissues, by culturing them in a medium "conditioned" by this normal tissue, or by RNA isolated from normal endoderm/mesoderm. Additionally, RNA isolated from normal anterior endoderm/mesoderm conditioned medium corrects the mutant hearts in a dose-dependent manner. A cDNA library is constructed using this RNA. On the basis of sequence analyses on this cDNA library, it was estimated that 56% of the total RNA present in the conditioned medium is rRNA, while 44% is nonribosomal RNA. One of the nonribosomal RNAs that showed no significant homology with other known sequences in the Genebank was examined further. An RT-PCR analysis showed that this RNA (designated "N1") is expressed in juvenile skeletal muscle, brain, and heart in significant amounts, less in the lung and not at all in the liver tissue. Affinity-purified polyclonal antipeptide antibodies were produced against the most antigenic portion of the polypeptide which was deduced from this RNA. Western blot analyses of adult heart homogenates, using these antibodies, showed a specific doublet staining at 67 kDa and 65 kDa. These doublets were purified and analyzed for their amino acid composition which showed that both bands most likely belong to the same protein. The N1-protein was further investigated to determine its localization in normal isolated hearts at embryonic stages 35, 38, and 41 and on cross-sections through the heart regions of whole normal embryos at stages 16, 33-34, 37-38, and 41-42 using immunohistochemical techniques and confocal microscopy. In addition, mutant embryos at stage 37-38 were studied for the presence and distribution of the N1-protein on cross-sections through their heart regions. The N1-protein staining was significantly reduced in mutant hearts when compared to normal.

Analysis of dissociation and unfolding of oligomeric proteins using a flat bed gel electrophoresis at high pressure.

A slab gel electrophoresis apparatus with the ability to operate over a pressure range of 10(-3) to 2 kbar is described. The system presented here is an improvement of a previous apparatus (A. A. Paladini, J. L. Silva, and G. Weber, Anal. Biochem. 161, 358-364, 1987). It consists of a flat bed gel, with a significantly enlarged buffer reservoir, which eliminates the requirement of high concentrations of running buffers, and at the same time allows shorter runs, leading to enhanced resolution and reproducibility. The application of the method to the dissociation of the tetramer glycogen phosphorylase a as a function of hydrostatic pressure is described. The flat geometry of the apparatus allows for the first time the analysis of the stability of oligomers and their constituent subunits to chemical denaturation by urea gradient electrophoresis gels at high pressure. Dimeric hexokinase shows a reversible cooperative unfolding transition with a midpoint at 3.8 M urea. In contrast, the monomers unfold at very low urea concentration (< 1.0 M). The observed differences in stability validates oligomerization as an important stabilizing element of the protein structure.

Melanotropin structure-activity studies on melanocytes of the teleost fish, Synbranchus marmoratus.

The minimal sequence of alpha-MSH required for full agonism on fish (Synbranchus marmoratus) melanocytes was determined to be Ac-alpha-MSH5-10-NH2 since Ac-alpha-MSH6-10-NH2 and Ac-alpha-MSH6-9-NH2 were inactive. The N-terminal tripeptide sequence, Ser-Tyr-Ser, lacked any contribution to potency since the 4-13 (Ac-[Nle4]-alpha-MSH4-13-NH2) sequence was equipotent to alpha-MSH. The important potentiating amino acids were found to be Met at position 4 of the amino terminus and Val at position 13 of the carboxy terminus of the hormone, since Ac-alpha-MSH4-10-NH2 was about 100 times more potent than the Ac-alpha-MSH5-10-NH2 sequence, and Ac-[Nle4]-alpha-MSH4-13-NH2 was about 10 times more active than Ac-[Nle4]-alpha-MSH4-12-NH2. The minimal sequence for equipotency to alpha-MSH was demonstrated to be Ac-[Nle4]-alpha-MSH4-13-NH2. [Nle4, D-Phe7]-alpha-MSH was about 10 times more active than alpha-MSH. Unexpectingly, several conformationally restricted cyclic melanotropins were either partial agonists ([Cys4, Cys10]-alpha-MSH) or totally inactive (Ac[Cys4, Cys10]-alpha-MSH4-10-NH2) on fish melanocytes. These results point out some rather remarkable differences between S. marmoratus and tetrapod melanophores relative to structural requirements for MSH receptor recognition and signal transduction.