[Show abstract][Hide abstract] ABSTRACT: BALB/cJ is a strain susceptible to stress and extremely susceptible to a defective hedonic impact in response to chronic stressors. The strain offers much promise as an animal model for the study of stress related disorders. We present a comparative hippocampal gene expression study on the effects of unpredictable chronic mild stress on BALB/cJ and C57BL/6J mice. Affymetrix MOE 430 was used to measure hippocampal gene expression from 16 animals of two different strains (BALB/cJ and C57BL/6J) of both sexes and subjected to either unpredictable chronic mild stress (UCMS) or no stress. Differences were statistically evaluated through supervised and unsupervised linear modelling and using Weighted Gene Coexpression Network Analysis (WGCNA). In order to gain further understanding into mechanisms related to stress response, we cross-validated our results with a parallel study from the GENDEP project using WGCNA in a meta-analysis design.
The effects of UCMS are visible through Principal Component Analysis which highlights the stress sensitivity of the BALB/cJ strain. A number of genes and gene networks related to stress response were uncovered including the Creb1 gene. WGCNA and pathway analysis revealed a gene network centered on Nfkb1. Results from the meta-analysis revealed a highly significant gene pathway centred on the Ubiquitin C (Ubc) gene. All pathways uncovered are associated with inflammation and immune response.
The study investigated the molecular mechanisms underlying the response to adverse environment in an animal model using a GxE design. Stress-related differences were visible at the genomic level through PCA analysis highlighting the high sensitivity of BALB/cJ animals to environmental stressors. Several candidate genes and gene networks reported are associated with inflammation and neurogenesis and could serve to inform candidate gene selection in human studies and provide additional insight into the pathology of Major Depressive Disorder.
[Show abstract][Hide abstract] ABSTRACT: AIMS Aspirin is widely used as an anti-platelet agent for cardiovascular prophylaxis. Despite aspirin treatment, many patients experience recurrent thrombotic events, and aspirin resistance may contribute to this. We examined the prevalence of aspirin resistance in a healthy population, and investigated whether the platelet proteome differed in aspirin-resistant subjects. METHODS Ninety-three healthy subjects received aspirin 300 mg daily for 28 days. Before and at the end of treatment, urine was taken to determine 11-dehydrothromboxane B-2, and blood was taken to measure arachidonic acid (AA)-induced aggregation of platelet-rich plasma and to interrogate the platelet proteome by mass spectrometric analysis with further confirmation of findings using Western blotting. RESULTS In two of the 93 subjects, neither AA-induced aggregation nor urinary 11-dehydrothromboxane B-2 was effectively suppressed by aspirin, despite measurable plasma salicylate concentrations, suggesting the presence of true aspirin resistance. Despite no detectable differences in the platelet proteome at baseline, following aspirin a marked increase was seen in platelet glycoprotein IIIa expression in the aspirin-resistant but not aspirin-sensitive subjects. An increase in platelet glycoprotein IIIa expression with aspirin resistance was confirmed in a separate cohort of 17 patients with stable coronary artery disease on long term aspirin treatment, four of whom exhibited aspirin resistance. CONCLUSIONS In a healthy population, true aspirin resistance is uncommon but exists. Resistance is associated with an increase in platelet glycoprotein IIIa expression in response to aspirin. These data shed new light on the mechanism of aspirin resistance, and provide the potential to identify aspirin-resistant subjects using a novel biomarker.
British Journal of Clinical Pharmacology 08/2014; 78(2):320-8. DOI:10.1111/bcp.12335 · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we present a pharmacoproteomic investigation of response to antidepressants two inbred strains. Our aim was to uncover molecular mechanisms underlying antidepressant action and identify new biomarkers to determine therapeutic response to two antidepressants with proven efficacy in the treatment of depression but divergent mechanisms of action. Mice were treated with the pro-noradrenergic drug nortriptyline, the pro-serotonergic drug escitalopram or saline. Quantitative proteomic analyses were undertaken on hippocampal tissue from a study design that used two inbred mouse strains, two depressogenic protocols and a control condition, (maternal separation, chronic mild stress, control), two antidepressant drugs and two dosing protocols. The proteomic analysis was aimed at the identification of specific drug-response markers. Complementary approaches, 2DE and isobaric tandem mass tagging (TMT), were applied to the selected experimental groups. To investigate the relationship between proteomic profiles, depressogenic protocols and drug response, 2DE and TMT data sets were analysed using multivariate methods. The results highlighted significant strain- and stress-related differences across both 2DE and TMT data sets and identified the three gene products involved in serotonergic (PXBD5, YHWAB, SLC25A4) and one in noradrenergic antidepressant action (PXBD6).
[Show abstract][Hide abstract] ABSTRACT: Peripheral biomarkers of Alzheimer's disease (AD) reflecting early neuropathological change are critical to the development of treatments for this condition. The most widely used indicator of AD pathology in life at present is neuroimaging evidence of brain atrophy. We therefore performed a proteomic analysis of plasma to derive biomarkers associated with brain atrophy in AD. Using gel based proteomics we previously identified seven plasma proteins that were significantly associated with hippocampal volume in a combined cohort of subjects with AD (N = 27) and MCI (N = 17). In the current report, we validated this finding in a large independent cohort of AD (N = 79), MCI (N = 88) and control (N = 95) subjects using alternative complementary methods-quantitative immunoassays for protein concentrations and estimation of pathology by whole brain volume. We confirmed that plasma concentrations of five proteins, together with age and sex, explained more than 35% of variance in whole brain volume in AD patients. These proteins are complement components C3 and C3a, complement factor-I, γ-fibrinogen and alpha-1-microglobulin. Our findings suggest that these plasma proteins are strong predictors of in vivo AD pathology. Moreover, these proteins are involved in complement activation and coagulation, providing further evidence for an intrinsic role of these pathways in AD pathogenesis.
PLoS ONE 12/2011; 6(12):e28527. DOI:10.1371/journal.pone.0028527 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of internal peptide standards in selected reaction monitoring experiments enables absolute quantitation. Here, we describe three approaches addressing calibration of peptide concentrations in complex matrices and assess their performance in terms of trueness and precision. The simplest approach described is single reference point quantitation where a heavy peptide is spiked into test samples and the endogenous analyte quantified relative to the heavy peptide internal standard. We refer to the second approach as normal curve quantitation. Here, a constant amount of heavy peptide and a varying amount of light peptide are spiked into matrix to construct a calibration curve. This accounts for matrix effects but due to the presence of endogenous analyte, it is usually not possible to determine the lower LOQ. We refer to the third method as reverse curve quantitation. Here, a constant amount of light peptide and a varying amount of heavy peptide are spiked into matrix to construct a calibration curve. Because there is no contribution to the heavy peptide signal from endogenous analyte, it is possible to measure the equivalent of a blank sample and determine LOQ. These approaches are applied to human plasma samples and used to assay peptides of a set of apolipoproteins.
[Show abstract][Hide abstract] ABSTRACT: Blood-based markers reflecting core pathological features of Alzheimer's disease (AD) in pre-symptomatic individuals are likely to accelerate the development of disease-modifying treatments. Our aim was to discover plasma proteins associated with brain amyloid-β (Aβ) burden in non-demented older individuals. We performed discovery-phase experiments using two dimensional gel electrophoresis (2DGE) and mass spectrometry-based proteomic analysis of plasma in combination with 11C-PiB PET imaging of the brain in samples collected 10 years prior to the PET scans. Confirmatory studies used ELISA assays in a separate set of blood samples obtained within a year of the PET scans. We observed that a panel of 18 2DGE plasma protein spots effectively discriminated between individuals with high and low brain Aβ. Mass spectrometry identified these proteins, many of which have established roles in Aβ clearance, including a strong signal from apolipoprotein-E (ApoE). In validation-phase studies, we observed a strong association between plasma ApoE concentration and Aβ burden in the medial temporal lobe. Targeted voxel-based analysis localized this association to the hippocampus and entorhinal cortex. APOE ε4 carriers also showed greater Aβ levels in several brain regions relative to ε4 non-carriers. These results suggest that both peripheral concentration of ApoE protein and APOE genotype are related to early neuropathological changes in brain regions vulnerable to AD pathology even in the non-demented elderly. Our strategy combining proteomics with in vivo brain amyloid imaging holds promise for the discovery of biologically relevant peripheral markers in those at risk for AD.
[Show abstract][Hide abstract] ABSTRACT: Blood-based analytes may be indicators of pathological processes in Alzheimer disease (AD).
To identify plasma proteins associated with AD pathology using a combined proteomic and neuroimaging approach.
Discovery-phase proteomics to identify plasma proteins associated with correlates of AD pathology. Confirmation and validation using immunodetection in a replication set and an animal model.
A multicenter European study (AddNeuroMed) and the Baltimore Longitudinal Study of Aging.
Patients with AD, subjects with mild cognitive impairment, and healthy controls with standardized clinical assessments and structural neuroimaging.
Association of plasma proteins with brain atrophy, disease severity, and rate of clinical progression. Extension studies in humans and transgenic mice tested the association between plasma proteins and brain amyloid.
Clusterin/apolipoprotein J was associated with atrophy of the entorhinal cortex, baseline disease severity, and rapid clinical progression in AD. Increased plasma concentration of clusterin was predictive of greater fibrillar amyloid-beta burden in the medial temporal lobe. Subjects with AD had increased clusterin messenger RNA in blood, but there was no effect of single-nucleotide polymorphisms in the gene encoding clusterin with gene or protein expression. APP/PS1 transgenic mice showed increased plasma clusterin, age-dependent increase in brain clusterin, as well as amyloid and clusterin colocalization in plaques.
These results demonstrate an important role of clusterin in the pathogenesis of AD and suggest that alterations in amyloid chaperone proteins may be a biologically relevant peripheral signature of AD.
Archives of general psychiatry 07/2010; 67(7):739-48. DOI:10.1001/archgenpsychiatry.2010.78 · 13.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Quantitative proteomics using isobaric labeling typically involves sample digestion, peptide-level labeling and 2D LC-MS/MS. Proteomic analysis of complex samples can potentially be performed more comprehensively with GeLC-MS/MS. However, combining this approach with peptide-level labeling of multiple in-gel digests from entirely sectioned gel lanes can introduce many points of variation and adversely affect the final quantitative accuracy. Alternatively, samples labeled with isobaric tags at the protein level can be combined and analyzed by GeLC-MS/MS as a single gel lane. A caveat to this strategy is that only lysine residues are labeled, which might limit protein digestion and quantitation of peptides. Here we have compared a protein-level labeling GeLC-MS/MS strategy with a peptide-level labeling 2D LC-MS/MS approach, using mouse hippocampus synaptosomes and isobaric tandem mass tags. Protein-level labeling enabled the identification of 3 times more proteins (697 versus 241) than did peptide-level labeling, and importantly for quantitation, twice as many proteins with labeled peptides (480 versus 232) were identified. Preliminary in silico analysis also suggested the alternative use of Asp-N to trypsin to circumvent the interference of lysine labeling on protein digestion. Use of Asp-N resulted in the effective analysis of fewer peptides than with trypsin for the protein-level approach (1677 versus 3131), but yielded a similar quantitative proteomic coverage in terms of both peptides (1150 versus 1181) and proteins (448 versus 480). Taken together, these experiments demonstrate that protein-level labeling combined with GeLC-MS/MS is an effective strategy for the multiplexed quantitation of synaptosomal preparations, and may also be applicable to samples of a similar proteomic complexity and dynamic range of protein abundance.
Journal of Proteome Research 03/2010; 9(5):2725-33. DOI:10.1021/pr900627e · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is a progressive neurodegenerative disorder where definite diagnosis can only be made postmortem, and for which the most promising peripheral markers of disease state and severity have been found in the cerebrospinal fluid. However, recent results suggest that differences in the levels of certain plasma proteins do exist between AD patients and non-demented controls (NDC). Herein, we undertook an untargeted discovery study using isobaric mass tagging to compare the plasma protein levels between slow cognitive declining AD patients, rapid cognitive declining AD patients (RCD) and NDC subjects. Subsequent relative quantification and statistical analysis identified a list of candidate proteins able to distinguish RCD from NDC groups based on multivariate analysis. Selected proteins were then validated by western blot analysis in an independent sample set of 60 AD and 35 NDC subjects. In this cohort, AD patients displayed significantly lower plasma gelsolin levels compared to NDC subjects. Additionally, gelsolin levels correlated with disease progression rate estimated by Mini-Mental Status Examination decline per year. In order to further investigate gelsolin expression, three different brain regions from an additional cohort of 23 subjects and their respective plasma samples were analysed. No significant change in brain gelsolin levels could be established between AD and control subjects. Interestingly, this study reveals yet another condition where plasma gelsolin levels are decreased and our findings, together with the reported interaction of gelsolin and amyloid-beta, makes plasma gelsolin an attractive candidate for further studies targeted at better understanding disease progression in AD.
[Show abstract][Hide abstract] ABSTRACT: Biomarkers of brain disorders are urgently needed to aid diagnosis, monitor disease progression, and, as new medicines are introduced, detect the patient's response to treatment. Proteomics provides the opportunity to discover novel biochemical markers based on protein or peptide changes, either in concentration levels or post-translational modification status. There are many challenges associated with proteomics studies, and this article represents a review of the issues discussed during the proteomics breakout sessions held at the Biomarkers for Brain Disorders conference in Oxford in January 2009. Although to date, there are very few qualified biomarkers that have arisen as a result of proteomics efforts, we remain optimistic that proteomics will deliver biomarkers for brain disorders. To be successful, we need to recognize that such endeavors are likely to require multidisciplinary teams and continued collaboration between academia, the biotechnology industry, and the pharmaceutical sector.
Annals of the New York Academy of Sciences 10/2009; 1180(1):68-74. DOI:10.1111/j.1749-6632.2009.05018.x · 4.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tandem Mass Tags (TMT) are suited to both global and targeted quantitation approaches of proteins and peptides. Different versions of these tags allow for the generation of both isobaric and isotopic sets of reagents sharing the same common structure. This feature allows for a straightforward transfer of data obtained during discovery studies into targeted investigations. In prior discovery studies, an isobaric set of these reagents was used to identify Neisseria meningitidis proteins expressed under iron-limitation. Here, we apply isotopic versions of those reagents in combination with single reaction monitoring to verify selected candidates found to be differentially regulated in these discovery studies, representing both well-known and novel iron-regulated proteins, such as the MtrCDE drug efflux pump. In this targeted approach (TMT-SRM), the selectivity of SRM is maintained while allowing the incorporation of an internal reference standard into the experiment. By monitoring 184 transitions, TMT-SRM resulted in the quantitation of 33 peptides representing 12 proteins. The acquired data corroborated the results obtained during the discovery phase. Furthermore, these data obtained by MS-based quantitation of peptides were independently confirmed by western blotting results, an orthogonal approach based on quantitation at the protein level.
Journal of proteomics 09/2009; 73(2):231-9. DOI:10.1016/j.jprot.2009.09.002 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel strategy consisting of cleavable Isotope-Coded Affinity Tag (cICAT) combined with MASCOT Distiller was evaluated as a tool for the quantification of proteins in "abnormal" patient plasma, prepared by pooling samples from patients with acute stroke. Quantification of all light and heavy cICAT-labelled peptide ion pairs was obtained using MASCOT Distiller combined with a proprietary software. Peptides displaying differences were selected for identification by MS. These preliminary results show the promise of our approach to identify potential biomarkers.