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1.  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
2.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  Site-specific proteomic analysis of lipoxidation adducts in cardiac mitochondria reveals chemical diversity of 2-alkenal adduction 
Journal of proteomics  2011;74(11):2417-2429.
The modification of proteins by lipid peroxidation products has been linked to numerous diseases and age-related disorders. Here we report on the identification of endogenous protein targets of electrophilic 2-alkenals in cardiac mitochondria. An aldehyde/keto-specific chemical labeling and affinity strategy in combination with LC-MS/MS resulted in 39 unique lipoxidation sites on 27 proteins. Several of the target sites were modified by a variety of 2-alkenal products including acrolein, β-hydroxyacrolein, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and 4-oxo-2-nonenal. Many of the adduction sites are implicated in the catalytic function of key mitochondrial enzymes suggesting potential impact on pathways and overall mitochondrial function.
PMCID: PMC3199298  PMID: 21513823
Carbonyls; aldehyde-reactive probe; mitochondria; proteomics; lipid peroxidation product; acrolein; hydroxynonenal
17.  Tandem mass spectrometric characterization of thiol peptides modified by the chemoselective cationic sulfhydryl reagent (4-iodobutyl)triphenylphosphonium 
Fixed charge chemical modifications on peptides and proteins can impact the fragmentation behaviors in tandem mass spectrometry (MS/MS). In this study, we employed a thiol-specific cationic alkylation reagent, (4-iodobutyl)triphenylphosphonium (IBTP), to selectively modify cysteine thiol groups in mitochondrial proteome samples. Tandem mass spectrometric characteristics of butyltriphenylphosphonium (BTP)-modified peptides were evaluated by comparison to their carbamidomethylated (CAM) analogues using a quadrupole time-of-flight (Q-TOF) instrument under low energy collision-induced dissociation (CID) conditions. Introduction of the fixed charge modification resulted in the observation of peptide and fragment (bn and yn) ions with higher charge states than those observed for CAM-modified analogues. The charged BTP moiety had a significant effect on the neighboring amide bond fragmentation products. A decrease in relative abundances of the product ions at the corresponding cleavage sites was observed compared to those from the CAM-modified derivative. This effect was particularly noticeable when an Xxx-Pro bond was in the vicinity of a BTP group. We hypothesized that the presence of a phosphonium moiety will reduce the tendency for protonation of the proximal amide bonds in the peptide backbone. Indeed, calculations indicated that proton affinities of backbone amide bonds close to the modified cysteine residues were generally 20-50 kcal/mol lower for BTP-modified peptides then for the unmodified or CAM-modified analogues with the sequence motif -Ala-Cys-Alan-Ala2-, -Ala-Cys-Alan-Pro-Ala-, and -Ala-Pro-Alan-Cys-Ala-, n = 0-3. (220 words)
PMCID: PMC3187551  PMID: 21952891
Peptide fragmentation; fixed charge modification; thiol-specific modification; proline effect
18.  Measuring Copper and Zinc Superoxide Dismutase from Spinal Cord Tissue using Electrospray Mass Spectrometry 
Analytical biochemistry  2011;415(1):52-58.
Metals are key cofactors for many proteins, yet quantifying the metals bound to specific proteins is a persistent challenge in vivo. We have developed a rapid and sensitive method using electrospray ionization mass spectrometry to measure Cu,Zn superoxide dismutase (SOD1) directly from the spinal cord of SOD1-overexpressing transgenic rats. Metal dyshomeostasis has been implicated in motor neuron death in amyotrophic lateral sclerosis (ALS). Using the assay, SOD1 was directly measured from 100 μg of spinal cord, allowing for anatomical quantitation of apo, metal-deficient, and holo SOD1. SOD1 was bound on a C4 ZipTip® that served as a disposable column, removing interference by physiological salts and lipids. SOD1 was eluted with 30% acetonitrile plus 100 μM formic acid to provide sufficient hydrogen ions to ionize the protein without dislodging metals. SOD1 was quantified by including bovine SOD1 as an internal standard. SOD1 could be measured in subpicomole amounts and resolved to within two Daltons of the predicted parent mass. The methods can be adapted to quantify modifications to other proteins in vivo that can be resolved by mass spectrometry.
PMCID: PMC3118564  PMID: 21453670
Metals; Superoxide dismutase; Amyotrophic Lateral Sclerosis; Mass Spectrometry; Copper; Zinc
19.  Conformational studies of the robust 2-Cys peroxiredoxin Salmonella typhimurium AhpC by solution phase hydrogen/deuterium (H/D) exchange monitored by electrospray ionization mass spectrometry 
This is the first comprehensive HX-MS study of a “robust” 2-Cys peroxiredoxin (Prx), namely Salmonella typhimurium AhpC (StAhpC). Prx proteins control intracellular peroxide levels and are abundant antioxidant proteins in eukaryotes, archaea and bacteria. Crystal structural analyses and structure/activity studies of several bacterial and mammalian 2-Cys Prxs have revealed that the activity of 2-Cys Prxs is regulated by redox-dependent oligmerization and a sensitivity of the active site cysteine residue to overoxidation. The propensity to overoxidation is linked to the conformational flexibility of the peroxidatic active site loop. The HX-MS results emphasize the modulation of the conformational motility of the active site loop by disulfide formation. To obtain information on the conformational impact of decamer formation on the active site loop motility, mutants with Thr77 substituted by Ile, a decamer-disrupting mutation or by Val, a decamer-stabilizing mutation, were studied. For the isoleucine mutant, enhanced mobility was observed for regions encompassing the α4 helix located in the dimer-dimer interface and regions surrounding the peroxidatic loop. In contrast, the T77V mutation resulted in an increase in conformational stability in most regions of the protein except for the active site loop and the region encompassing the resolving cysteine.
PMCID: PMC3079231  PMID: 21516234
mass spectrometry; hydrogen exchange; deuterium; conformation; folding; peroxiredoxins
20.  A targeted mass spectrometry-based approach for the identification and characterization of proteins containing α-aminoadipic and γ-glutamic semialdehyde residues 
Analytical and bioanalytical chemistry  2010;398(7-8):2905-2914.
The site-specific identification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues in proteins is reported. Semialdehydic protein modifications result from the metal-catalyzed oxidation of Lys or Arg and Pro residues, respectively. Most of the analytical methods for the analysis of protein carbonylation measure change to the global level of carbonylation and fail to provide details regarding protein identity, site, and chemical nature of the carbonylation. In this work, we used a targeted approach, which combines chemical labeling, enrichment, and tandem mass spectrometric analysis, for the site-specific identification of AAS and GGS sites in proteins. The approach is applied to in vitro oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and an untreated biological sample, namely cardiac mitochondrial proteins. The analysis of GAPDH resulted in the site-specific identification of two AAA and four GGS residues. Computational evaluation of the identified AAS and GGS sites in GAPDH indicated that these sites are located in flexible regions, show high solvent accessibility values, and are in proximity with possible metal ion binding sites. The targeted proteomic analysis of semialdehydic modifications in cardiac mitochondria yielded nine AAS modification sites which were unambiguously assigned to distinct lysine residues in the following proteins: ATP/ATP translocase isoforms 1 and 2, ubiquinol cytochrome-c reductase core protein 2, and ATP synthase α-subunit.
PMCID: PMC3071856  PMID: 20957471
Aldehyde-reactive probe; Protein carbonyls; Mass spectrometry; Metal-catalyzed oxidation; α-Aminoadipic semialdehyde; γ-Glutamic semialdehyde
21.  Site-specific protein adducts of 4-hydroxy-2(E)-nonenal in human THP-1 monocytic cells: Protein carbonylation is diminished by ascorbic acid 
The protein targets and sites of modification by 4-hydroxy-2(E)-nonenal (HNE) in human monocytic THP-1 cells after exogenous exposure to HNE were examined using a multi-pronged proteomic approach involving electrophoretic, immunoblotting and mass spectrometric methods. Immunoblot analysis using monoclonal anti-HNE antibodies showed several proteins as targets of HNE adduction. Pretreatment of THP-1 cells with ascorbic acid resulted in reduced levels of HNE-protein adducts. Biotinylation of Michael-type HNE adducts using an aldehyde-reactive hydroxylamine-functionalized probe (aldehyde-reactive probe, ARP) and subsequent enrichment facilitated the identification and site-specific assignment of the modifications by LC-MS/MS analysis. Sixteen proteins were unequivocally identified as targets of HNE adduction and eighteen sites of HNE modification at Cys and His residues were assigned. HNE exposure of THP-1 cells resulted in the modification of proteins involved in cytoskeleton organization and regulation, proteins associated with stress responses and enzymes of the glycolytic and other metabolic pathways. This study yielded the first evidence of site-specific adduction of HNE to Cys-295 in tubulin α-1B chain, Cys-351 and Cys-499 in α-actinin-4, Cys-328 in vimentin, Cys-369 in D-3-phosphoglycerate dehydrogenase and His-246 in aldolase A.
PMCID: PMC2825578  PMID: 20043646
THP-1; HNE; ascorbic acid; aldehyde-reactive probe; HNE-protein adducts; tandem mass spectrometry
22.  Supercomplexes of the Mitochondrial Electron Transport Chain Decline in the Aging Rat Heart 
Accumulation of mitochondrial electron transport chain (ETC) defects is a recognized hallmark of the age-associated decline in cardiac bioenergetics; however, the molecular events involved are only poorly understood. In the present work, we hypothesized that age-related ETC deterioration stemmed partly from disassociation of large solid-state macromolecular assemblies termed “supercomplexes”. Mitochondrial proteins from young and old rat hearts were separated by Blue Native-PAGE, protein bands analyzed by LC-MALDI-MS/MS, and protein levels quantified by densitometry. Results showed that supercomplexes comprised of various stoichiometries of complexes I, III and IV were observed, and declined significantly (p < 0.05, n = 4) with age. Supercomplexes displaying the highest molecular masses were the most severely affected. Considering that certain diseases (e.g. Barth Syndrome) display similar supercomplex destabilization as our results for aging, the deterioration in ETC supercomplexes may be an important underlying factor for both impaired mitochondrial function and loss of cardiac bioenergetics with age.
PMCID: PMC2762268  PMID: 19679098
aging; rat heart mitochondria; electron transport supercomplexes; blue native-polyacrylamide gel electrophoresis
23.  The developmental impact of prenatal stress, prenatal dexamethasone and postnatal social stress on physiology, behaviour and neuroanatomy of primate offspring: studies in rhesus macaque and common marmoset 
Psychopharmacology  2010;214(1):33-53.
Exposure of the immature mammalian brain to stress factors, including stress levels of glucocorticoids, either prenatally or postnatally, is regarded as a major regulatory factor in short- and long-term brain function and, in human, as a major aetiological factor in neuropsychiatric disorders. Experimental human studies are not feasible and animal studies are required to demonstrate causality and elucidate mechanisms. A number of studies have been conducted and reviewed in rodents but there are relatively few studies in primates.
Here we present an overview of our published studies and some original data on the effects of: (1) prenatal stress on hypothalamic–pituitary–adrenal (HPA) re/activity and hippocampus neuroanatomy in juvenile-adolescent rhesus macaques; (2) prenatal dexamethasone (DEX) on HPA activity, behaviour and prefrontal cortex neuroanatomy in infant-adolescent common marmosets; (3) postnatal daily parental separation stress on HPA re/activity, behaviour, sleep and hippocampus and prefrontal cortex neuroanatomy in infant-adolescent common marmoset.
Prenatal stress increased basal cortisol levels and reduced neurogenesis in macaque. Prenatal DEX was without effect on HPA activity and reduced social play and skilled motor behaviour in marmoset. Postnatal social stress increased basal cortisol levels, reduced social play, increased awakening and reduced hippocampal glucocorticoid and mineralocorticoid receptor expression in marmoset.
Perinatal stress-related environmental events exert short- and long-term effects on HPA function, behaviour and brain status in rhesus macaque and common marmoset. The mechanisms mediating the enduring effects remain to be elucidated, with candidates including increased basal HPA function and epigenetic programming.
PMCID: PMC3045510  PMID: 20809212
Glucocorticoid receptor; Hippocampus; HPA axis; Mineralocorticoid receptor; Neurogenesis; Neuropsychiatric disorders; Prefrontal cortex; Skilled motor behaviour; Sleep; Social play
24.  Hop proanthocyanidins induce apoptosis, protein carbonylation, and cytoskeleton disorganization in human colorectal adenocarcinoma cells via reactive oxygen species 
Proanthocyanidins (PCs) have been shown to suppress the growth of diverse human cancer cells and are considered as promising additions to the arsenal of chemopreventive phytochemicals. An oligomeric mixture of PCs from hops (Humulus lupulus) significantly decreased cell viability of human colon cancer HT-29 cells in a dose-dependent manner. Hop PCs, at 50 or 100 μg/ml, exhibited apoptosis-inducing properties as shown by the increase in caspase-3 activity. Increased levels of intracellular reactive oxygen species (ROS) was accompanied by an augmented accumulation of protein carbonyls. Mass spectrometry-based proteomic analysis in combination with 2-alkenal-specific immunochemical detection identified β-actin and protein disulfide isomerase as major putative targets of acrolein adduction. Incubation of HT-29 cells with hop PCs resulted in morphological changes that indicated disruption of the actin cytoskeleton. PC-mediated hydrogen peroxide (H2O2) formation in the cell culture media was also quantified; but, the measured H2O2 levels would not explain the observed changes in the oxidative modifications of actin. These findings suggest new modes of action for proanthocyandins as antitumorgenic agents in human colon cancer cells, namely, promotion of protein oxidative modifications and cytoskeleton derangement.
PMCID: PMC2888903  PMID: 19271284
Acrolein; actin; apoptosis; proanthocyanidins; protein carbonyls; HT-29 cells
Brain research  2007;1176:133-142.
Exposure to rotenone, a widely used pesticide, has been suggested to increase the risk of developing Parkinson’s disease. Studies indicate that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species. The present work was conducted to determine to what extent (−)-epigallocatechin-3-gallate (EGCG), a widely-used dietary supplement, modulates the cytotoxicity of rotenone in human neuroblastoma SH-SY5Y cells. Our results indicate that EGCG shows concentration-dependent effects on ROS production and cytotoxicity in SH-SY5Y cells. Treatment of these dopaminergic cells with rotenone (1–50 μM) alone or EGCG (25 or 50 μM) alone caused a significant decrease in cell viability. Pretreatment of SH-SY5Y cells with 25 or 50 μM EGCG potentiated the cytotoxicity of rotenone. The exacerbating effect of EGCG on rotenone toxicity may involve an apoptotic mechanism as shown by the enhancement of caspase-3 activity and activation of other caspases in rotenone-treated SH-SY5Y cells. The potentiating effect of EGCG on rotenone toxicity may be attributed to the enhanced production of intracellular superoxide in SH-SY5Y cells. The enhanced intracellular production of ROS by rotenone-EGCG combination may also account for the increased formation of protein carbonyls in 10,000 × g fraction of SH-SY5Y cells detected by anti-HNE antibody. For instance, core histones and nuclear ribonuclear proteins were identified as major putative in vivo targets of HNE. Our present findings indicate that more detailed mechanistic studies are necessary to fully understand the chemistry of EGCG and to justify its use as potentially health-promoting dietary supplement, e.g. in the prevention of neurodegenerative diseases associated with oxidative stress.
PMCID: PMC2104474  PMID: 17900545
Parkinson’s disease; neuroblastoma cells; SH-SY5Y cells; rotenone; EGCG; superoxide; protein carbonyls

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