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1.  Assessment of global proteome in LNCaP cells by 2D-RP/RP LC-MS/MS following sulforaphane exposure 
EuPA open proteomics  2015;9:34-40.
The phytochemical sulforaphane can induce cell cycle arrest and apoptosis in metastatic prostate cancer cells, though the mechanism of action is not fully known. We conducted a global proteome analysis in LNCaP metastatic prostate cancer cells to characterize how global protein signature responds to sulforaphane. We conducted parallel analyses to evaluate semi-quantitative 1-dimensional versus 2-dimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) and their utility in characterizing whole cell lysate. We show that 2-dimensional LC-MS/MS can be a useful tool for characterizing global protein profiles and identify TRIAP1 as a novel regulator of cell proliferation in LNCaP metastatic prostate cancer cells.
Graphical Abstract
PMCID: PMC4666544  PMID: 26640761
prostate cancer; sulforaphane; mass spectrometry
2.  HDAC6 activity is not required for basal autophagic flux in metastatic prostate cancer cells 
Experimental Biology and Medicine  2015;241(11):1177-1185.
Histone deacetylase 6 is a multifunctional lysine deacetylase that is recently emerging as a central facilitator of response to stress and may play an important role in cancer cell proliferation. The histone deacetylase 6-inhibitor tubacin has been shown to slow the growth of metastatic prostate cancer cells and sensitize cancer cells to chemotherapeutic agents. However, the proteins histone deacetylase 6 interacts with, and thus its role in cancer cells, remains poorly characterized. Histone deacetylase 6 deacetylase activity has recently been shown to be required for efficient basal autophagic flux. Autophagy is often dysregulated in cancer cells and may confer stress resistance and allow for cell maintenance and a high proliferation rate. Tubacin may therefore slow cancer cell proliferation by decreasing autophagic flux. We characterized the histone deacetylase 6-interacting proteins in LNCaP metastatic prostate cancer cells and found that histone deacetylase 6 interacts with proteins involved in several cellular processes, including autophagy. Based on our interaction screen, we assessed the impact of the histone deacetylase 6-inhibitor tubacin on autophagic flux in two metastatic prostate cancer cell lines and found that tubacin does not influence autophagic flux. Histone deacetylase 6 therefore influences cell proliferation through an autophagy-independent mechanism.
PMCID: PMC4950308  PMID: 26643866
Autophagy; metastatic prostate cancer; HDAC6; tubacin
3.  Analysis of autophagic flux in response to sulforaphane in metastatic prostate cancer cells 
Molecular nutrition & food research  2015;59(10):1954-1961.
The phytochemical sulforaphane has been shown to decrease prostate cancer metastases in a genetic mouse model of prostate carcinogenesis, though the mechanism of action is not fully known. Sulforaphane has been reported to stimulate autophagy, and modulation of autophagy has been proposed to influence sulforaphane cytotoxicity; however, no conclusions about autophagy can be drawn without assessing autophagic flux, which has not been characterized in prostate cancer cells following sulforaphane treatment.
Methods and Results
We conducted an investigation to assess the impact of sulforaphane on autophagic flux in two metastatic prostate cancer cell lines at a concentration shown to decrease metastasis in vivo. Autophagic flux was assessed by multiple autophagy related proteins and substrates. We found that sulforaphane can stimulate autophagic flux and cell death only at high concentrations, above what has been observed in vivo.
These results suggest that sulforaphane does not directly stimulate autophagy or cell death in metastatic prostate cancer cells under physiologically relevant conditions, but instead supports the involvement of in vivo factors as important effectors of sulforaphane- mediated prostate cancer suppression.
PMCID: PMC4651621  PMID: 26108801
autophagy; cancer; flux; prostate; sulforaphane
4.  Descriptive, cross-country analysis of the nurse practitioner workforce in six countries: size, growth, physician substitution potential 
BMJ Open  2016;6(9):e011901.
Many countries are facing provider shortages and imbalances in primary care or are projecting shortfalls for the future, triggered by the rise in chronic diseases and multimorbidity. In order to assess the potential of nurse practitioners (NPs) in expanding access, we analysed the size, annual growth (2005–2015) and the extent of advanced practice of NPs in 6 Organisation for Economic Cooperation and Development (OECD) countries.
Cross-country data analysis of national nursing registries, regulatory bodies, statistical offices data as well as OECD health workforce and population data, plus literature scoping review.
NP and physician workforces in 6 OECD countries (Australia, Canada, Ireland, the Netherlands, New Zealand and USA).
Primary and secondary outcome measures
The main outcomes were the absolute and relative number of NPs per 100 000 population compared with the nursing and physician workforces, the compound annual growth rates, annual and median percentage changes from 2005 to 2015 and a synthesis of the literature on the extent of advanced clinical practice measured by physician substitution effect.
The USA showed the highest absolute number of NPs and rate per population (40.5 per 100 000 population), followed by the Netherlands (12.6), Canada (9.8), Australia (4.4), and Ireland and New Zealand (3.1, respectively). Annual growth rates were high in all countries, ranging from annual compound rates of 6.1% in the USA to 27.8% in the Netherlands. Growth rates were between three and nine times higher compared with physicians. Finally, the empirical studies emanating from the literature scoping review suggested that NPs are able to provide 67–93% of all primary care services, yet, based on limited evidence.
NPs are a rapidly growing workforce with high levels of advanced practice potential in primary care. Workforce monitoring based on accurate data is critical to inform educational capacity and workforce planning.
PMCID: PMC5020757  PMID: 27601498
nursing; health workforce; physicians; PRIMARY CARE; nurse practitioner
5.  Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102 
BMC Genomics  2016;17:457.
Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture.
The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90.
Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2738-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4907049  PMID: 27296936
Anabaena; Anatoxin-a; Synteny; Cyanobacteria; Mobilome; Long-read sequencing; PacBio; Illumina; Tandem repeat; Structural variation
6.  18O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes 
PLoS ONE  2016;11(6):e0157118.
The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to uncover changes in cellular metabolism that are not detectable by time-resolved metabolomics.
PMCID: PMC4898700  PMID: 27275782
7.  Expanding clinical roles for nurses to realign the global health workforce with population needs: a commentary 
Many countries, including Israel, face health workforce challenges to meet the needs of their citizens, as chronic conditions increase. Provider shortages and geographical maldistribution are common. Increasing the contribution of nurse practitioners and other advanced practice nursing roles through task-shifting and expansion of scope-of-practice can improve access to care and result in greater workforce efficiency. Israel and many other countries are introducing reforms to expand nurses’ scope-of-practice. Recent international research offers three policy lessons for how countries just beginning to implement reforms could bypass policy barriers to implementation. First, there is substantial evidence on the equivalence in quality of care, patient safety and high consumer acceptance which should move policy debates from if to how to effectively implement new roles in practice. Second, regulatory and finance policies as well as accessible advanced education are essential to facilitate realignment of roles. Third, country experience suggests that advanced practice roles for nurses improve the attractiveness of nursing as a career thus contributing to solving nursing shortages rather than exacerbating them. Designing enabling policy environments and removing barriers will gain in relevance in the future as the demand for high-quality, patient-centered care is increasing.
PMCID: PMC4897947  PMID: 27280014
8.  A Multiplex Biomarker Approach for the Diagnosis of Transitional Cell Carcinoma from Canine Urine 
Analytical biochemistry  2014;455:41-47.
Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a non-invasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.
PMCID: PMC4874471  PMID: 24704347
Canine; Biomarkers; Liquid chromatography; Tandem mass spectrometry; Transitional cell carcinoma
9.  Label-Free Proteomics Assisted by Affinity Enrichment for Elucidating the Chemical Reactivity of the Liver Mitochondrial Proteome toward Adduction by the Lipid Electrophile 4-hydroxy-2-nonenal (HNE) 
The analysis of oxidative stress-induced post-translational modifications remains challenging due to the chemical diversity of these modifications, the possibility of the presence of positional isomers and the low stoichiometry of the modified proteins present in a cell or tissue proteome. Alcoholic liver disease (ALD) is a multifactorial disease in which mitochondrial dysfunction and oxidative stress have been identified as being critically involved in the progression of the disease from steatosis to cirrhosis. Ethanol metabolism leads to increased levels of reactive oxygen species (ROS), glutathione depletion and lipid peroxidation. Posttranslational modification of proteins by electrophilic products of lipid peroxidation has been associated with governing redox-associated signaling mechanisms, but also as contributing to protein dysfunction leading to organelle and liver injury. In particular the prototypical α,β-unsaturated aldehyde, 4-hydroxy-2-nonenal (HNE), has been extensively studied as marker of increased oxidative stress in hepatocytes. In this study, we combined a LC-MS label-free quantification method and affinity enrichment to assess the dose-dependent insult by HNE on the proteome of rat liver mitochondria. We used a carbonyl-selective probe, the ARP probe, to label HNE-protein adducts and to perform affinity capture at the protein level. Using LC-MS to obtain protein abundance estimates, a list of protein targets was obtained with increasing concentration of HNE used in the exposure studies. In parallel, we performed affinity capture at the peptide level to acquire site-specific information. Examining the concentration-dependence of the protein modifications, we observed distinct reactivity profiles for HNE-protein adduction. Pathway analysis indicated that proteins associated with metabolic processes, including amino acid, fatty acid, and glyoxylate and dicarboxylate metabolism, bile acid synthesis and TCA cycle, showed enhanced reactivity to HNE adduction. Whereas, proteins associated with oxidative phosphorylation displayed retardation toward HNE adduction. We provide a list of 31 protein targets with a total of 61 modification sites that may guide future targeted LC-MS assays to monitor disease progression and/or intervention in preclinical models of ALD and possibly other liver diseases with an oxidative stress component.
PMCID: PMC4865762  PMID: 27242993
protein carbonylation; 4-hydroxy-2-nonenal; Michael adducts; aldehyde-reactive probe; mitochondria; liver; oxidative stress; Alcoholic liver disease
10.  Conformational dynamics of human FXR-LBD ligand interactions studied by hydrogen/deuterium exchange mass spectrometry: Insights into the antagonism of the hypolipidemic agent Z-guggulsterone 
Biochimica et biophysica acta  2014;1844(9):1684-1693.
Farnesoid X Receptor (FXR) is a member of the nuclear receptor superfamily of transcription factors that plays a key role in the regulation of bile acids, lipid and glucose metabolisms. The regulative function of FXR is governed by conformational changes of the ligand binding domain (LBD) upon ligand binding. Although FXR is a highly researched potential therapeutic target, only a limited number of FXR-agonist complexes have been successfully crystallized and subsequently yielded high resolution structures. There is currently no structural information of any FXR-antagonist complexes publically available. We therefore explored the use of amide hydrogen/deuterium exchange (HDX) coupled with mass spectrometry for characterizing conformational changes in the FXR-LBD upon ligand binding. Ligand-specific deuterium incorporation profiles were obtained for three FXR ligand chemotypes: GW4064, a synthetic non-steroidal high affinity agonist; the bile acid chenodeoxycholic acid (CDCA), the endogenous low affinity agonist of FXR; and Z-guggulsterone (GG), an in vitro antagonist of the steroid chemotype. Comparison of the HDX profiles of their ligand-bound FXR-LBD complexes revealed a unique mode of interaction for GG. The conformational features of the FXR-LBD-antagonist interaction are discussed.
PMCID: PMC4137978  PMID: 24953769
Farnesoid X receptor; hydrogen/deuterium exchange; mass spectrometry; conformation; dynamics; ligand interaction; guggulsterone
11.  Proteome-Driven Elucidation of Adaptive Responses to Combined Vitamin E and C Deficiency in Zebrafish 
Journal of Proteome Research  2014;13(3):1647-1656.
The purpose of this study was to determine the system-wide consequences of deficiencies in two essential micronutrients, vitamins E and C, on the proteome using zebrafish (Danio rerio) as one of the few vertebrate models that similar to humans cannot synthesize vitamin C. We describe a label-free proteomics workflow to detect changes in protein abundance estimates dependent on vitamin regimes. We used ion-mobility-enhanced data-independent tandem mass spectrometry to determine differential regulation of proteins in response to low dietary levels of vitamin C with or without vitamin E. The detection limit of the method was as low as 20 amol, and the dynamic range was five orders of magnitude for the protein-level estimates. On the basis of the quantitative changes obtained, we built a network of protein interactions that reflect the whole organism’s response to vitamin C deficiency. The proteomics-driven study revealed that in vitamin-E-deficient fish, vitamin C deficiency is associated with induction of stress response, astrogliosis, and a shift from glycolysis to glutaminolysis as an alternative mechanism to satisfy cellular energy requirements.
PMCID: PMC3993953  PMID: 24476500
vitamin C; vitamin E; ion mobility; MSE; label-free quantification; zebrafish; glycolysis; glutaminolysis
12.  Application of Paper Strip Extraction in Combination with LC–MS-MS in Pharmacokinetics 
Spectroscopy (Springfield, Or.)  2013;39(10):s18-s25.
Recent advances in sampling techniques in the pharmaceutical industry sparked significant interest in applying improvements to extraction methods for greater analyte detection and quantitation. In particular, the dried blood spot (DBS) sampling technique has numerous advantages compared to traditional methods such as liquid–liquid extraction, including the use of small sample volumes, less sample processing, and less exposure to toxic solvents (ether, methyl tert-butyl ether [MTBE], and dichloromethane). In this article, we discuss the adaptation of DBS technology to develop and validate a novel paper strip extraction method for the analysis of natural product metabolites in biological samples obtained from a human pharmacokinetic study of xanthohumol, a hop prenylflavonoid.
PMCID: PMC4201118  PMID: 25328281
Methods in enzymology  2010;473:305-330.
This chapter describes a mass spectrometry-based strategy that facilitates the unambiguous identification and characterization of proteins modified by lipid peroxidation-derived 2-alkenals. The approach employs a biotinylated hydroxyl amine derivative as an aldehyde/keto reactive probe in conjunction with selective enrichment and tandem mass spectrometric analysis. Methodological details are given for model studies involving a distinct protein and 4-hydroxy-2-nonenal (HNE). The method was also evaluated for an exposure study of a cell culture system with HNE that yielded the major protein targets of HNE in human monocytic THP-1 cells. The application of the approach to complex biological systems is demonstrated for the identification and characterization of endogenous protein targets of aldehydic lipid peroxidation products present in cardiac mitochondria.
PMCID: PMC4191911  PMID: 20513485
14.  A freshwater cyanophage whose genome indicates close relationships to photosynthetic marine cyanomyophages 
Environmental microbiology  2011;13(7):1858-1874.
Bacteriophage S-CRM01 has been isolated from a freshwater strain of Synechococcus and shown to be present in the upper Klamath River valley in northern California and Oregon. The genome of this lytic T4-like phage has a 178,563 bp circular genetic map with 297 predicted protein-coding genes and 33 tRNA genes that represent all 20 amino acid specificities. Analyses based on gene sequence and gene content indicate a close phylogenetic relationship to the “photosynthetic” marine cyanomyophages infecting Synechococcus and Prochlorococcus. Such relatedness suggests that freshwater and marine phages can draw on a common gene pool. The genome can be considered as being comprised of three regions. Region 1 is populated predominantly with structural genes, recognized as such by homology to other T4-like phages and by identification in a proteomic analysis of purified virions. Region 2 contains most of the genes with roles in replication, recombination, nucleotide metabolism and regulation of gene expression, as well as 5 of the 6 signature genes of the photosynthetic cyanomyophages (hli03, hsp20, mazG, phoH and psbA; cobS is present in Region 3). Much of Regions 1 and 2 are syntenous with marine cyanomyophage genomes, except that a segment encompassing Region 2 is inverted. Region 3 contains a high proportion (85%) of genes that are unique to S-CRM01, as well as most of the tRNA genes. Regions 1 and 2 contain many predicted late promoters, with a combination of CTAAATA and ATAAATA core sequences. Two predicted genes that are unusual in phage genomes are homologs of cellular spoT and nusG.
PMCID: PMC4185292  PMID: 21605306
Myoviridae; Klamath River; cyanomyophage; freshwater phage; nusG; spoT; phage tRNA; T4-like phage
15.  Effects of low and high dose intraarticular tiludronate on synovial fluid and clinical variables in healthy horses—a preliminary investigation 
PeerJ  2014;2:e534.
To determine effects of intraarticularly administered tiludronate on articular cartilage in vivo, eight healthy horses were injected once with tiludronate (low dose tiludronate [LDT] 0.017 mg, n = 4; high dose tiludronate [HDT] 50 mg, n = 4) into one middle carpal joint and with saline into the contralateral joint. Arthrocentesis of both middle carpal joints was performed pre-treatment, and 10 min, 24 h, 48 h, 7 and 14 days after treatment. Synovial nucleated cell counts and total solids, tiludronate, sulfated glycosaminoglycan (sGAG), chondroitin sulfate 846 epitope (CS-846, a measure of aggrecan synthesis), and collagen type II cleavage neoepitope (C2C) concentrations were determined. Histologic analysis of joint tissues and sGAG quantitation in cartilage was performed at 14 days in HDT horses. Data were analyzed by repeated measures non-parametric ANOVA and Wilcoxon signed-rank test. High dose tiludronate administration produced synovial fluid tiludronate concentrations of 2,677,500 ng/mL, exceeding concentrations that were safe for cartilage in vitro, and LDT administration produced synovial fluid concentrations of 1,353 ng/mL, remaining below concentrations considered potentially detrimental to cartilage. With HDT, synovial fluid total solids concentration was higher at 24 h and 7 days and sGAG concentration was higher at 48 h, compared to control joints. Synovial fluid CS-846 concentration was increased over pre-treatment values in HDT control but not in HDT treated joints at 24 and 48 h. All joints (HDT and LDT control and treated) showed a temporary decrease in synovial fluid C2C concentration, compared to pre-treatment values. Histologic features of articular cartilage and synovial membrane did not differ between HDT treated and control joints. High dose tiludronate treatment caused a transient increase in synovial total solids and temporarily increased proteoglycan degradation in cartilage. Although clinical significance of these changes are questionable, as they did not result in articular cartilage damage, further investigation of the safety of intraarticular HDT in a larger number of horses is warranted.
PMCID: PMC4157236  PMID: 25237596
Cartilage; Horse; Tiludronate; Bisphosphonate; Joint injection; Glycosaminoglycans
16.  Protein modifications by electrophilic lipoxidation products: Adduct formation, chemical strategies and tandem mass spectrometry for their detection and identification 
Mass spectrometry reviews  2014;33(3):157-182.
The post-translational modification of proteins by electrophilic oxylipids is emerging as an important mechanism that contributes to the complexity of proteomes. Enzymatic and nonenzymatic oxidation of biological lipids results in the formation of chemically diverse electrophilic carbonyl compounds, such as 2-alkenals and 4-hydroxy alkenals, epoxides and eicosanoids with reactive cyclopentenone structures. These lipoxidation products are capable of modifying proteins. Originally considered solely as markers of oxidative insult, more recently the modifications of proteins by lipid peroxidation products are being recognized as a new mechanism of cell signaling with relevance to redox homeostasis, adaptive response and inflammatory resolution. The growing interest in protein modifications by reactive oxylipid species necessitates the availability of methods that are capable of detecting, identifying and characterizing these protein adducts in biological samples with high complexity. However, the efficient analysis of these chemically diverse proteins presents a considerable analytical challenge. We first provide an introduction into the chemistry and biological relevance of the protein adduction by electrophilic lipoxidation products. We then provide an overview of tandem mass spectrometry approaches that have been developed in recent years for the interrogation of protein modifications by electrophilic oxylipid species.
PMCID: PMC4138024  PMID: 24818247
HSAB theory; electrophilic lipoxidation products; HNE; aldehyde-reactive probe; collision induced dissociation; electron capture dissociation; electron transfer dissociation; ion mobility mass spectrometry
17.  A proteome-driven elucidation of adaptive responses to combined vitamin E and C deficiency in zebrafish 
Journal of proteome research  2014;13(3):1647-1656.
The purpose of this study was to determine the system-wide consequences of deficiencies in two essential micronutrients, vitamin C and E, on the proteome using zebrafish (Danio rerio) as one of the few vertebrate models that similar to humans cannot synthesize vitamin C. We describe a label-free proteomics workflow to detect changes in protein abundance estimates dependent on vitamin regimes. We used ion mobility-enhanced data-independent tandem mass spectrometry to determine differential regulation of proteins in response to low dietary levels of vitamin C with or without vitamin E. The detection limit of the method was as low as 20 amol and the dynamic range was 5 orders of magnitude for the protein level estimates. Based on the quantitative changes obtained, we built a network of protein interactions that reflects the whole organism’s response to vitamin C deficiency. The proteomics-driven study revealed that in vitamin E deficient fish, vitamin C deficiency is associated with induction of stress response, astrogliosis, and a shift from glycolysis to glutaminolysis as an alternative mechanism to satisfy cellular energy requirements.
PMCID: PMC3993953  PMID: 24476500
Vitamin C; ascorbic acid; ion mobility; MSE; label free quantification; zebrafish; glycolysis; glutaminolysis
18.  Simultaneous, untargeted metabolic profiling of polar and non-polar metabolites by LC-Q-TOF mass spectrometry 
At its most ambitious, untargeted metabolomics aims to characterize and quantify all of the metabolites in any system. Metabolites are often present at a broad range of concentrations and possess diverse physical properties complicating this task. Performing multiple sample extractions, concentrating sample extracts, and using several separation and detection methods are common strategies to overcome these challenges but require a great amount of resources. This protocol describes the untargeted, metabolic profiling of polar and non-polar metabolites with a single extraction and using a single analytical platform.
PMCID: PMC3707319  PMID: 23670865
Untargeted metabolomics; LC-MS/MS; hypothesis generation
19.  Caffeoylquinic acids in Centella asiatica protect against β-amyloid toxicity 
The accumulation of β-amyloid (Aβ) is a hallmark of Alzheimer’s disease and is known to result in neurotoxicity both in vivo and in vitro. We previously demonstrated that treatment with the water extract of Centella asiatica (CAW) improves learning and memory deficits in Tg2576 mice, an animal model of Aβ accumulation. However the active compounds in CAW remain unknown. Here we used two in vitro models of Aβ toxicity to confirm this neuroprotective effect, and identify several active constituents of the CAW extract. CAW reduced Aβ-induced cell death and attenuated Aβ-induced changes in tau expression and phosphorylation in both the MC65 and SH-SY5Y neuroblastoma cell lines. We confirmed and quantified the presence of several mono- and dicaffeoylquinic acids (CQAs) in CAW using chromatographic separation coupled to mass spectrometry and ultraviolet spectroscopy. Multiple dicaffeoylquinic acids showed efficacy in protecting MC65 cells against Aβ-induced cytotoxicity. Isochlorogenic acid A and 1,5-dicaffeoylquinic acid were found to be the most abundant CQAs in CAW, and the most active in protecting MC65 cells from Aβ-induced cell death. Both compounds showed neuroprotective activity in MC65 and SH-SY5Y cells at concentrations comparable to their levels in CAW. Each compound not only mitigated Aβ-induced cell death, but was able to attenuate Aβ-induced alterations in tau expression and phosphorylation in both cell lines, as seen with CAW. These data suggest that CQAs are active neuroprotective components in CAW, and therefore are important markers for future studies on CAW standardization, bioavailability and dosing.
PMCID: PMC3973491  PMID: 24448790
β-amyloid toxicity; Centella asiatica; caffeoylquinic acids; tau; neuroprotection
20.  LCP 140° Pediatric Hip Plate for fixation of proximal femoral valgisation osteotomy 
Femoral osteotomy is one of the most widely performed reconstructive operations in pediatric orthopedic surgery. Many implants for fixation have been used, but so far there is no literature about the application and outcome of the LCP 140° Pediatric Hip Plate for proximal femoral valgisation in children.
Data of patients with a valgisation of the proximal femur using the LCP 140° Pediatric Hip Plate between February 2011 and July 2012 were retrospectively collected and analyzed.
We included 10 patients (11 hips) with a mean follow-up of 15.3 ± 6.3 months (range 5.6–23 months). The mean age was 9.6 ± 1.2 years (range 7.3–11.8 years) with a mean hospital stay of 5.2 ± 1.7 days (range 3–9 days). Callus formation was observed in all cases at 6 weeks postoperative control and consolidation was shown after a mean time of 14.1 ± 2.3 weeks (range 12.1–19.1 weeks). There was no delayed union or any case of non-union in our series. The stability of the operative reduction including the corrected neck-shaft angle (mean 19° ± 7.9°; range 10.5°–38.5°) was maintained during the follow-up period. No cases of recurrence (varisation) or complications requiring further treatment or revision were observed.
In our series, the 140° LCP Pediatric Hip Plate was shown to be safe and applicable in the clinical setting with good results. We therefore consider this device to be valuable for the correction of pathologic varus conditions of the proximal femur in children.
PMCID: PMC3935024  PMID: 24488843
Femoral osteotomy; Valgisation; Pediatric Hip Plate
21.  Surface-exposed proteins of pathogenic mycobacteria and the role of cu-zn superoxide dismutase in macrophages and neutrophil survival 
Proteome Science  2013;11:45.
Pathogenic mycobacteria are important agents causing human disease. Mycobacterium avium subsp. hominissuis (M. avium) is a species of recalcitrant environmental pathogen. The bacterium forms robust biofilms that allow it to colonize and persist in austere environments, such as residential and commercial water systems. M. avium is also an opportunistic pathogen that is a significant source of mortality for immune-compromised individuals. Proteins exposed at the bacterial surface play a central role in mediating the relationship between the bacterium and its environment. The processes underlying both biofilm formation and pathogenesis are directly dependent on this essential subset of the bacterial proteome. Therefore, the characterization of the surface-exposed proteome is an important step towards an improved understanding of the mycobacterial biology and pathogenesis. Here we examined the complement of surface exposed proteins from Mycobacterium avium 104, a clinical isolate and reference strain of Mycobacterium avium subsp. hominissuis. To profile the surface-exposed proteins of viable M. avium 104, bacteria were covalently labeled with a membrane impermeable biotinylation reagent and labeled proteins were affinity purified via the biotin-streptavidin interaction. The results provide a helpful snapshot of the surface-exposed proteome of this frequently utilized reference strain of M. avium. A Cu-Zn SOD knockout mutant, MAV_2043, a surface identified protein, was evaluated regarding its role in the survival in both macrophages and neutrophils.
PMCID: PMC4176128  PMID: 24283571
Mycobacterium avium; Surface-exposed proteome; Shotgun proteomics; Cu-Zn SOD
22.  A Comparative `Bottom Up' Proteomics Strategy for the Site-specific Identification and Quantification of Protein Modifications By Electrophilic Lipids 
Journal of proteomics  2012;75(18):5724-5733.
We report a mass spectrometry-based comparative “bottom up” proteomics approach that combines d0/d4-succinic anhydride labeling with commercially available hydrazine (Hz)-functionalized beads (Affi-gel Hz beads) for detection, identification and relative quantification of site-specific oxylipid modifications in biological matrices. We evaluated and applied this robust and simple method for the quantitative analysis of oxylipid protein conjugates in cardiac mitochondrial proteome samples isolated from 3- and 24-month-old rat hearts. The use of d0/d4-succinic anhydride labeling, Hz-bead based affinity enrichment, nanoLC fractionation and MALDI-ToF/ToF tandem mass spectrometry yielded relative quantification of oxylipid conjugates with residue-specific modification information. Conjugation of acrolein (ACR), 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-noneal (ONE) to cysteine, histidine and lysine residues were identified. HHE conjugates were the predominant subset of Michael-type adducts detected in this study. The HHE conjugates showed higher levels in mitochondrial preparations from young heart congruent with previous findings by others that the n-3/n-6 PUFA ratio is higher in young heart mitochondrial membranes. Although this study focuses on protein adducts of reactive oxylipids the method might be equally applicable to protein carbonyl modifications caused by metal catalyzed oxidation reactions.
PMCID: PMC3468693  PMID: 22842153
Protein carbonyls; oxidative stress; lipid peroxidation products; mitochondria; mass spectrometry; heart
23.  Electrospray Quadrupole Travelling Wave Ion Mobility Time-of-Flight Mass Spectrometry for the Detection of Plasma Metabolome Changes Caused by Xanthohumol in Obese Zucker (fa/fa) Rats 
Metabolites  2013;3(3):701-717.
This study reports on the use of traveling wave ion mobility quadrupole time-of-flight (ToF) mass spectrometry for plasma metabolomics. Plasma metabolite profiles of obese Zucker fa/fa rats were obtained after the administration of different oral doses of Xanthohumol; a hop-derived dietary supplement. Liquid chromatography coupled data independent tandem mass spectrometry (LC-MSE) and LC-ion mobility spectrometry (IMS)-MSE acquisitions were conducted in both positive and negative modes using a Synapt G2 High Definition Mass Spectrometry (HDMS) instrument. This method provides identification of metabolite classes in rat plasma using parallel alternating low energy and high energy collision spectral acquisition modes. Data sets were analyzed using pattern recognition methods. Statistically significant (p < 0.05 and fold change (FC) threshold > 1.5) features were selected to identify the up-/down-regulated metabolite classes. Ion mobility data visualized using drift scope software provided a graphical read-out of differences in metabolite classes.
PMCID: PMC3901285  PMID: 24958146
obesity; xanthohumol; travelling wave ion mobility; mass spectrometry; lipidomics
24.  Mass spectrometry-based quantification of myocardial protein adducts with acrolein in an in vivo model of oxidative stress 
Molecular nutrition & food research  2011;55(9):1401-1410.
Acrolein exposure leads to the formation of protein-acrolein adducts. Protein modification by acrolein has been associated with various chronic diseases including cardiovascular and neurodegenerative diseases. Here we report an analytical strategy that enables the quantification of Michael-type protein adducts of acrolein in mitochondrial proteome samples using liquid chromatography in combination with tandem mass spectrometry and selected ion monitoring (LC-MS/MS SRM) analysis. Our approach combines site-specific identification and relative quantification at the peptide level of protein–acrolein adducts in relation to the unmodified protein thiol pool. Treatment of 3-month old rats with CCl4, an established in vivo model of acute oxidative stress, resulted in significant increases in the ratios of distinct acrolein-adducted peptides to the corresponding unmodified thiol-peptides obtained from proteins that were isolated from cardiac mitochondria. The mitochondrial proteins that were found adducted by acrolein were malate dehydrogenase, NADH dehydrogenase [ubiquinone] flavoprotein 1, cytochrome c oxidase subunit VIb isoform 1, ATP synthase d chain, and ADP/ATP translocase 1. The findings indicate that protein modification by acrolein has potential value as an index of mitochondrial oxidative stress.
PMCID: PMC3517132  PMID: 21809440
Acrolein; protein carbonyls; aldehyde/keto-reactive probe; mitochondria; selected reaction monitoring
25.  Surface Proteome of “Mycobacterium avium subsp. hominissuis” during the Early Stages of Macrophage Infection 
Infection and Immunity  2012;80(5):1868-1880.
“Mycobacterium avium subsp. hominissuis” is a robust and pervasive environmental bacterium that can cause opportunistic infections in humans. The bacterium overcomes the host immune response and is capable of surviving and replicating within host macrophages. Little is known about the bacterial mechanisms that facilitate these processes, but it can be expected that surface-exposed proteins play an important role. In this study, the selective biotinylation of surface-exposed proteins, streptavidin affinity purification, and shotgun mass spectrometry were used to characterize the surface-exposed proteome of M. avium subsp. hominissuis. This analysis detected more than 100 proteins exposed at the bacterial surface of M. avium subsp. hominissuis. Comparisons of surface-exposed proteins between conditions simulating early infection identified several groups of proteins whose presence on the bacterial surface was either constitutive or appeared to be unique to specific culture conditions. This proteomic profile facilitates an improved understanding of M. avium subsp. hominissuis and how it establishes infection. Additionally, surface-exposed proteins are excellent targets for the host adaptive immune system, and their identification can inform the development of novel treatments, diagnostic tools, and vaccines for mycobacterial disease.
PMCID: PMC3347459  PMID: 22392927

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