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Previous studies suggest that microglial-expressed Apolipoprotein E (ApoE) is necessary to shift microglia into a neurodegenerative transcriptional state in Alzheimer's disease (AD) mouse models. On the other hand, elimination of microglia shifts amyloid beta (Aβ) accumulation from parenchymal plaques to cerebral amyloid angiopathy (CAA), mimicking the effects of global APOE*4 knock-in. Here, we specifically knock-out microglial-expressed ApoE while keeping astrocytic-expressed ApoE intact. When microglial-specific ApoE is knocked-out of a 5xFAD mouse model of AD, we found a ~35% increase in average Aβ plaque size, but no changes in plaque load, microglial number, microglial clustering around Aβ plaques, nor the formation of CAA. Immunostaining revealed ApoE protein present in plaque-associated microglia in 5xFAD mice with microglial-specific ApoE knockout, suggesting that microglia can take up ApoE from other cellular sources. Mice with Apoe knocked-out of microglia had lower synaptic protein levels than control mice, indicating that microglial-expressed ApoE may have a role in synapse maintenance. Surprisingly, microglial-specific ApoE knock-out resulted in few differentially expressed genes in both 5xFAD and control mice; however, some rescue of 5xFAD associated neuronal networks may occur with microglial-specific ApoE knock-out as shown by weighted gene co-expression analysis. Altogether, our data indicates that microglial-expressed ApoE may not be necessary for plaque formation or for the microglial transcriptional shift into a Disease Associated Microglia state that is associated with reactivity to plaques but may be necessary for plaque homeostasis in disease and synaptic maintenance under normal conditions.

The Eu3+/2+ redox couple provides a convenient design platform for responsive pO2 sensors for magnetic resonance imaging (MRI). Specifically the Eu2+ ion provides T1w contrast enhancement under hypoxic conditions in tissues, whereas, under normoxia, the Eu3+ ion can produce contrast from chemical exchange saturation transfer in MRI. The oxidative stability of the Eu3+/2+ redox couple for a series of tetraaza macrocyclic complexes was investigated in this work using cyclic voltammetry. A series of Eu-containing cyclen-based macrocyclic complexes revealed positive shifts in the Eu3+/2+ redox potentials with each replacement of a carboxylate coordinating arm of the ligand scaffold with glycinamide pendant arms. The data obtained reveal that the complex containing four glycinamide coordinating pendant arms has the highest oxidative stability of the series investigated.

Microglia mediate chronic neuroinflammation following central nervous system (CNS) disease or injury, and in doing so, damage the local brain environment by impairing recovery and contributing to disease processes. Microglia are critically dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R) and can be eliminated via administration of CSF1R inhibitors. Resolving chronic neuroinflammation represents a universal goal for CNS disorders, but long-term microglial elimination may not be amenable to clinical use. Notably, withdrawal of CSF1R inhibitors stimulates new microglia to fully repopulate the CNS, affording an opportunity to renew this cellular compartment. To that end, we have explored the effects of acute microglial elimination, followed by microglial repopulation, in a mouse model of extensive neuronal loss. Neuronal loss leads to a prolonged neuroinflammatory response, characterized by the presence of swollen microglia expressing CD68 and CD45, as well as elevated levels of cytokines, chemokines, complement, and other inflammatory signals. These collective responses are largely resolved by microglial repopulation. Furthermore, microglial repopulation promotes functional recovery in mice, with elevated plus maze performance matching that of uninjured mice, despite the loss of 80% of hippocampal neurons. Analyses of synaptic surrogates revealed increases in PSD95 and synaptophysin puncta with microglial repopulation, suggesting that these cells sculpt and regulate the synaptic landscape. Thus, our results show that short-term microglial elimination followed by repopulation may represent a clinically feasible and novel approach to resolve neuroinflammatory events and promote brain recovery.

Alzheimer's disease (AD) is a major cause of dementia, disability, and death in the elderly. Despite recent advances in our understanding of the basic biological mechanisms underlying AD, we do not know how to prevent it, nor do we have an approved disease‐modifying intervention. Both are essential to slow or stop the growth in dementia prevalence. While our current animal models of AD have provided novel insights into AD disease mechanisms, thus far, they have not been successfully used to predict the effectiveness of therapies that have moved into AD clinical trials. The Model Organism Development and Evaluation for Late‐onset Alzheimer's Disease (MODEL‐AD; www.model-ad.org) Consortium was established to maximize human datasets to identify putative variants, genes, and biomarkers for AD; to generate, characterize, and validate the next generation of mouse models of AD; and to develop a preclinical testing pipeline. MODEL‐AD is a collaboration among Indiana University (IU); The Jackson Laboratory (JAX); University of Pittsburgh School of Medicine (Pitt); Sage BioNetworks (Sage); and the University of California, Irvine (UCI) that will generate new AD modeling processes and pipelines, data resources, research results, standardized protocols, and models that will be shared through JAX's and Sage's proven dissemination pipelines with the National Institute on Aging–supported AD Centers, academic and medical research centers, research institutions, and the pharmaceutical industry worldwide.

Diamagnetic chemical exchange saturation transfer (CEST) agents and paramagnetic CEST (PARACEST) agents use proton spin saturation and water exchange to produce exogenous-based contrast in MR images (1,2). Water protons bound to the inner sphere of a paramagnetic lanthanide ion (Ln ≠ La, Gd, or Lu) are typically shifted well away from the resonance frequency of bulk water protons (3). Radiofrequency saturation at the bound water frequency can produce indirect partial saturation of the bulk water signal through chemical exchange, resulting in negative contrast (i.e., darkening) in regions where the PARACEST agent is present. The percent reduction in bulk water signal is a function of agent concentration, radiofrequency saturation power and duration, and water exchange rate. Paramagnetic complexes can act as PARACEST agents if the water molecule exchange rate is slow when compared with the frequency difference between the two water pools (kex << Δω) (1,4). One potential advantage of PARACEST agents over Gd-based T1 agents is that image contrast can be turned on and off by inserting a frequency-selective radiofrequency saturation pulse before the imaging sequence. This frequency selection creates the potential of imaging multiple lanthanide-based agents simultaneously, each responding to a different physiological parameter or biochemical event. Such agents hold great potential to further extend the functional and molecular imaging capabilities of MR (5). Some published applications of PARACEST include measures of tissue pH (6,7), Zn2+ ion concentration (8), beta-cell function (9,10), and the tissue distribution of glucose (11–13). During initial in vivo studies of Eu3+-based PARACEST agents in mice, we observed a significant loss (>50%) of MR signal in the kidneys (after intravenous injection) and tumors grown from human cancer cell xenografts (after intratumoral injection). This signal loss was present even before a frequency-selective radiofrequency saturation pulse was applied and appeared to be caused by a local decrease in water T2 because of the presence of the PARACEST agent. Similar phenomena in mouse kidneys were recently observed using a Tm3+-based PARACEST agent and the OPARACHEE (on-resonance paramagnetic agent chemical exchange effect) pulse sequence (14,15). We also observed that this effect was more pronounced when using polymerized versions of the Eu3+-based agents that had higher local concentrations of Eu3+ ions and thus more water exchange sites per molecule (16). As the Eu3+ ion in these complexes has a relatively small paramagnetic relaxation enhancement when compared with other lanthanide ions (e.g., Gd3+, Tb3+, Dy3+ or Tm3+) (17), it was clear that the T2 line-broadening effect must originate with some other chemical feature of these complexes. This led to the hypothesis that the same slow-to-intermediate water exchange characteristics of these complexes that enable the PARACEST effect can also cause a local decrease in the bulk water signal though T2 exchange (T2exch). The effects of chemical exchange with diamagnetic molecules containing exchangeable —NH or —OH protons on transverse relaxation times have been known for over 40 years (18,19). Many early applications focused on using such molecules to suppress the strong water proton signal in high-resolution proton NMR experiments (20–24). Selective reduction of bulk water T2 by chemical exchange was first proposed as a method for MRI contrast by Aime et al. (25) and has recently been studied in vitro using the —NH-based proton exchange of iopamidol (26). In this article, we have extended this concept to weakly paramagnetic Eu3+ agents having variable water exchange rates. Both in vitro relaxation data and in vivo imaging data are presented along with simulations of T2exch using the Bloch equations modified for chemical exchange. It is shown that the transverse relaxivity (r2) of these agents is a function of water exchange kinetics and that r2 has a well-defined maximum at a specific exchange rate.

The oligomeric state and kinetics of ligand binding were measured for wild-type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N- and C-terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.

Aim To establish any differences between patients with and without a diagnosis of personality disorders, being treated in a secure inpatient service for offenders with intellectual disability (ID) in the UK. Method A cohort study involving a selected population of people with ID and offending behaviours. Results The study included a total of 138 patients, treated over a 6 year period – 77 with a dissocial or emotionally unstable personality disorder and 61 without. Women were more likely to be in the personality disorder group. Both groups had high prevalence of abuse with no significant differences. Depressive disorders and substance abuse were more common in the personality disorder group, while epilepsy and autistic spectrum disorders were more common in the non-personality disorder group. Rather than differences, what was more striking was the rate and range of these comorbidities across both groups. Although past histories of violence and institutional aggression were no different, compulsory detention under criminal sections and restriction orders were more common in the personality disorder group. There were no differences in treatment outcomes. Conclusions Although about half of patients detained in secure units for offenders with ID have a personality disorder, there were more similarities than differences between this group and the rest. While good treatment outcomes supported the case for specialised secure treatment units for people with ID, the case for establishing a more specialised ID–personality disorder unit was less convincing. There is also a need to explore whether there are alternative diagnostic models that can delineate better the group with personality difficulties in this population.

Magnetic resonance imaging (MRI) is one of the most versatile and powerful diagnostic tools in modern medicine. Recently, a conceptually different approach to contrast enhancement based on chemical exchange saturation transfer (CEST) has emerged that takes advantage of slow-to-intermediate exchange conditions between two or more pools of protons (kex ≤ Δω).[1] While the first reported CEST agents were diamagnetic molecules containing exchangeable NH and OH groups (Δω ≤ 5 ppm), it was later shown that the slow water exchange characteristics of certain paramagnetic Ln3+ complexes of DOTA-tetraamide ligands allows selective saturation of a hyperfine shifted Ln3+-bound water pool (Δω > 50 ppm) for creating CEST contrast.[2] Radio frequency (RF) saturation of highly shifted exchange resonances in paramagnetic systems offer significant advantages over diamagnetic CESTagents with small Δω values.[3]

The cause(s) of sporadic Alzheimer's disease (sAD) are complex and currently poorly understood. They likely result from a combination of genetic, environmental, proteomic and lipidomic factors that crucially occur only in the aged brain. Age-related changes in calcium levels and dynamics have the potential to increase the production and accumulation of both amyloid-beta peptide (Abeta) and tau pathologies in the AD brain, although these two pathologies themselves can induce calcium dyshomeostasis, particularly at synaptic membranes. This review discuses the evidence for a role for calcium dyshomeostasis in the initiation of pathology, as well as the evidence for these pathologies themselves disrupting normal calcium homeostasis, which lead to synaptic and neuronal dysfunction, synaptotoxicity and neuronal loss, underlying the dementia associated with the disease.

The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry. Copyright (C) 2008 John Wiley & Sons, Ltd.

Use of aerial photograph interpretation (API) in resource inventory projects recently has increased, and this reflects benefits like established protocols, high spatial resolution, readily available photography, and limited cost. Application of API to quantify features of aquatic habitats used by fishes, like submerged aquatic vegetation (SAV), has been advocated for decades but a paucity of use suggests inadequate awareness of the methods. This article reviews a protocol that guides the use of API to quantify features of aquatic habitats, and then uses examples from contrasting habitats in the Lake Ontario watershed from 1972–2003 to illustrate this protocol. Even though we used photographs originally collected for other purposes, API identified the change in minimum area and depth distribution of SAV over time. These observations reinforce how API can contribute information to resource inventories, and why investigators should expand use of API in studies of aquatic ecosystems.

Use of aerial photograph interpretation (API) in resource inventory projects recently has increased, and this reflects benefits like established protocols, high spatial resolution, readily available photography, and limited cost. Application of API to quantify features of aquatic habitats used by fishes, like submerged aquatic vegetation (SAV), has been advocated for decades but a paucity of use suggests inadequate awareness of the methods. This article reviews a protocol that guides the use of API to quantify features of aquatic habitats, and then uses examples from contrasting habitats in the Lake Ontario watershed from 1972–2003 to illustrate this protocol. Even though we used photographs originally collected for other purposes, API identified the change in minimum area and depth distribution of SAV over time. These observations reinforce how API can contribute information to resource inventories, and why investigators should expand use of API in studies of aquatic ecosystems.

Background The majority of variant and abnormal haemoglobins are clinically silent but may not be biochemically silent when it comes to HbA1c estimation. Case report We describe several cases in which there were problems in the determination of HbA1c for monitoring diabetes in patients that would not normally be classified as being at risk. Four of these cases were sufficiently unusual to warrant individual publication but all came from a very restricted geographical area with a population of only approximately 500 000. The significance of this is not that variant haemoglobins affect HbA1c analysis but that the occurrence of unexpected/unusual variant haemoglobins may be more frequent than most clinicians would expect, considering that only a small proportion of the basal population are diabetic and have their HbA1c monitored. Differences will exist between different areas due to differences in both the prevalence of variants and the analytical methods employed. Conclusions Consequently, we propose greater interaction between diabetologists and the laboratory in an attempt to identify these clinically but not biochemically silent variants to achieve a true estimation of the glycaemic control in affected patients.

Chronic hypoxia (CH) alters Ca2+ homeostasis in various cells and may contribute to disturbed Ca2+ homeostasis of Alzheimer's disease. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate the mechanism underlying augmentation of Ca2+ signalling by chronic hypoxia in type I cortical astrocytes. Application of bradykinin evoked significantly larger rises of [Ca2+]i in hypoxic cells as compared with control cells. This augmentation was prevented fully by either melatonin (150 micro m) or ascorbic acid (200 micro m), indicating the involvement of reactive oxygen species. Given the association between hypoxia and increased production of amyloid beta peptides (AbetaPs) of Alzheimer's disease, we performed immunofluorescence studies to show that hypoxia caused a marked and consistent increased staining for AbetaPs and presenilin-1 (PS-1). Western blot experiments also confirmed that hypoxia increased PS-1 protein levels. Hypoxic increases of AbetaP production was prevented with inhibitors of either gamma- or beta-secretase. These inhibitors also partially prevented the augmentation of Ca2+ signalling in astrocytes. Our results indicate that chronic hypoxia enhances agonist-evoked rises of [Ca2+]i in cortical astrocytes, and that this can be prevented by antioxidants and appears to be associated with increased AbetaP formation.

Alzheimer's disease (AD) is more prevalent following an ischemic or hypoxic episode, such as stroke. Indeed, brain levels of amyloid precursor protein (APP) and the cytotoxic amyloid beta peptide (Abeta) fragment are enhanced in these patients and in animal models following experimental ischaemia. We have investigated the effect of chronic hypoxia (CH; 2.5% O2, 24 h) on processing of APP in the human neuroblastoma, SH-SY5Y. We demonstrate that constitutive and muscarinic-receptor-enhanced secretion of the alpha-secretase cleaved fragment of APP, sAPPalpha, was reduced by approximately 60% in CH cells. The caspase inhibitor BOC-D(Ome)FMK did not reverse this effect of CH, and CH cells were as viable as controls, based on MTT assays. Thus, loss of sAPPalpha is not related to cell death or caspase processing of APP. Pre-incubation with antioxidants did not reverse the effect of CH, and the effect could not be mimicked by H2O2, discounting the involvement of reactive oxygen species in hypoxic loss of sAPPalpha. CH did not affect muscarinic activation of extracellular-signal regulated kinase. However, expression of ADAM 10 (widely believed to be alpha-secretase) was decreased approximately 50% following CH. Thus, CH selectively decreases processing of APP by the alpha-secretase pathway, most likely by decreasing levels of ADAM 10.

Exposure of PC12 cells to chronic hypoxia (CH; 10 % O(2), 24 h) augments catecholamine secretion via formation of a Cd2+-resistant Ca2+ influx pathway, and up-regulates native L-type Ca2+ channels. These effects are mimicked by exposure of cells to Alzheimer's disease-associated amyloid beta peptides (AbetaPs). Since pathological effects of AbetaPs have been associated with increased levels of reactive oxygen species (ROS), the involvement of ROS in hypoxia-mediated up-regulation of exocytosis and Ca2+ channel activity was examined. Both melatonin and ascorbic acid (two structurally unrelated antioxidants) fully blocked the enhancement of catecholamine secretion caused by CH (as determined amperometrically). Enhanced immunofluorescence, observed in chronically hypoxic cells using a primary monoclonal antibody raised against the N-terminus of AbetaP, was also suppressed by melatonin. Ascorbic acid, melatonin and ebselen (an additional antioxidant) also fully prevented augmentation of whole-cell Ca2+ currents caused by CH (as monitored using whole-cell patch-clamp recordings). Exposure of normoxic cells to H(2)O(2) (40 microM, 24 h), like hypoxia, caused Ca2+ channel up-regulation. Importantly, AbetaP formation appeared to be an absolute requirement for the effects of hypoxia, since the ability of CH to augment exocytosis and Ca2+ channel activity was blocked by two novel inhibitors of gamma secretase, an enzyme complex required for AbetaP formation. Our results indicate that the effects of hypoxia require ROS generation from AbetaPs, and suggest that elevated levels of ROS mediate hypoxic and AbetaP-mediated pathological remodelling of Ca2+ homeostasis.

Amyloid peptides (AbetaPs) are implicated in neuronal death associated with Alzheimer's disease. Their toxicity involves disruption cellular Ca(2+) homeostasis, leading to activation of caspases and cell death. Antioxidants can prevent such cell death and show beneficial clinical effects in Alzheimer's disease patients. Using the model neurosecretory cell line, PC12, we have shown that AbetaPs cause enhancement of evoked exocytosis via formation of a Cd(2+) -resistant Ca(2+) influx pathway, and also cause selective, functional up-regulation of current through L-type Ca(2+) channels. The involvement of reactive oxygen species (ROS) in these effects were investigated by examining the ability of various antioxidants to interfere with these responses. Both melatonin and ascorbic acid fully blocked the enhancement of catecholamine secretion caused by application of AbetaP((1-40)), as monitored in real time amperometrically, but inhibition of the transcriptional regulator NF-kappaB with SN-50 did not affect secretion. Enhanced immunofluorescence, observed in AbetaP-treated cells using a monoclonal antibody raised against the N-terminus of AbetaP, was also suppressed by melatonin. Ascorbic acid, melatonin and ebselen also fully prevented augmentation of whole-cell Ca(2+) currents caused by application of AbetaP((1-40)). By contrast, inhibitors of NF-kappaB (sulfasalazine and SN-50) were able to prevent AbetaP induced Ca(2+) channel current enhancement, whilst inhibitors of mitogen-activated protein kinase and protein kinase C could not. Our results indicate that augmentation or induction by AbetaPs of two important, distinct factors regulating Ca(2+) homeostasis is mediated by increased ROS production, but only one of these (up-regulation of native Ca(2+) channels) requires activation of NF-kappaB.

We developed a coating method to produce functionalized small quantum dots (sQDs), ~ 9 nm in diameter, that were stable for over a month. We made sQDs in four emission wavelengths, from 527 nm to 655 nm and with different functional groups. AMPA receptors on live neurons were labeled with sQDs and post-synaptic density proteins were visualized with super-resolution microscopy. Their diffusion behavior indicates that sQDs access the synaptic clefts significantly more often than commercial QDs.

Clinical studies indicate that neurodegeneration caused by Alzheimer's amyloid beta peptide (AbetaP) formation can be triggered or induced by prolonged (chronic) hypoxia. Here, we demonstrate that 24-h culture of PC12 cells in 10% O(2) leads to induction of a Cd(2+)-resistant Ca(2+) influx pathway and selective potentiation of L-type Ca(2+) current. Both effects were suppressed or prevented by a monoclonal antibody raised against the N'-terminus of AbetaP, and were fully mimicked by AbetaP(1-40 and) AbetaP(1-42), but not by AbetaP(40-1). Potentiation of L-type currents was also induced by exposure to AbetaP(25-35). Our results indicate that hypoxia induces enhancement of Ca(2+) channels, which is mediated by increased AbetaP formation.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.

Following previous analysis of the structure of Alvinella pompejana heaxagonal-bilayer haemoglobin (HBL Hb) [1], we report in this paper the structure of three other HBL Hbs belonging to Alvinella caudata, Paralvinella grasslei and Paralvinella palmiformis, members of the Alvinellidae, annelid family strictly endemic to deep-sea hydrothermal vents located on the ridge crests in the Pacific ocean. The multi-angle laser light scattering (MALLS) and fast protein liquid chromatography (FPLC) analysis revealed a broad range of molecular masses for the extracellular Hb molecules, 3517 ± 14 kDa (A. caudata), 3822 ± 28 kDa (P. grasslei) and 3750 ± 150 kDa (P. palmiformis). Native and derivative Hbs (reduced, carbamidomethylated and deglycosylated) were analysed by electrospray ionization mass spectroscopy (ESI-MS) and the data was processed by the maximum entropy deconvolution system (MaxEnt). The most important difference between alvinellid HBL Hbs was the variation in their composition, from two to four monomeric globin chains, and from one to four linker chains. Therefore, despite the fact that all these species belong to a single family, notable differences in the polypeptide chain composition of their HBL Hbs were observed, probably accounting for their different functional properties as previously reported by this group Toulmond, A., El Idrissi Slitine, F., De Frescheville, J. & Jouin, C. (1990) Biol. Bull.179, 366–373.

A systematic comparison between the up-front Collision induced dissociation (CID) mass spectra and low-energy CID tandem mass spectra from twenty-one singly and/or doubly charged peptides has been made. CID spectra of the peptides were recorded at different electrode voltages in the up-front source region of a single quadrupole instrument and different collision energies in the collision cell of a tandem quadrupole instrument. It was observed that up-front CID and low-energy CID yielded comparable product ion spectra from protonated peptides, and that the instrumental settings necessary for obtaining comparable CID mass spectra from the two methods are correlated.Chemicals/CAS: Peptide Fragments; Peptides

Branchipolynoe symmytilida and B. seepensis are two scaleworms (Polychaeta; Polynoidae) living commensally in the mantle cavity of deep-sea hydrothermal vent and cold-seep mussels. In contrast with littoral members of this family, the two species exhibit a large amount of extracellular hemoglobin (Hb) in their coelomic fluid. Gel filtration revealed the existence of four different Hbs: one minor, high molecular mass (3x10(6) Da) Hb, V1-Hb, reminiscent of a vascular hexagonal bilayer annelid Hb; two major coelomic Hbs, C1-Hb, and C2-Hb, with unusual masses for extracellular annelid Hbs of 153 and 124 kDa respectively; and a minor probably coelomic Hb of 23 kDa (C3-Hb). Using electrospray ionization mass spectrometry, SDS-PAGE after subtilisin treatment, and tandem mass spectrometry, we showed that C1-Hb is a trimer of a 57,996 Da chain and C2-Hb is a dimer of a 57,648 Da chain, each chain being a four-domain/four-heme polypeptide. This multimeric, multidomain arrangement is unique among annelid Hbs and appears different from that of other known multidomain Hbs.

The potential of electrospray mass spectrometry (ESMS) for the sequencing of glycopeptides was evaluated using quadrupole time-of-flight (QTOF) technology in the MS/MS mode. The location of O-glycosylation sites was possible in the positive ion (+) mode by detection of prominent y-and b-fragment ions from the underivatized TAP25-2 [T1APPAHGVT9S10APDT14RPAPGS20T21APPA], an overlapping sequence of MUC1 tandem repeats which had been glycosylated in vitro by two GalNAc residues in the positions T9 and T21. The high mass resolution and accuracy of QTOF-(+)ESMS allowed reliable structural assignments. The reduced complexity of the fragment spectra and the higher signal-to-noise ratio render QTOF-(+)ESMS an alternative mass spectrometric approach to the identification of O-glycosylation sites when compared with sequencing by post-source decay matrix-assisted laser desorption/ionization MS. Diagnostic ions from the N-terminus in the b-series offered direct evidence, which was supported by indirect evidence from the C-terminus ions of the y-series. The higher glycosylated GalNAc2-substituted fragments were mainly observed as multiply ionized species. © 1998 John Wiley & Sons, Ltd.