Milos V Novotny

Publications of Milos V Novotny

• Human ABCC1 interacts and colocalizes with ATP synthase α, revealed by interactive proteomics analysis.

  Authors: Youyun Yang, Zhaomin Li, Wei Mo, Raghuram Ambadipudi, Randy J Arnold, Petra Hrncirova, Milos V Novotny, Elias Georges, Jian-Ting Zhang

  Journal of proteome research. 12/2011; 11(2):1364-72.

  Human ABCC1 is a member of the ATP-binding cassette (ABC) transporter superfamily, and its overexpression has been shown to cause multidrug resistance by active efflux of a wide variety of anticancerHuman ABCC1 is a member of the ATP-binding cassette (ABC) transporter superfamily, and its overexpression has been shown to cause multidrug resistance by active efflux of a wide variety of anticancer drugs. ABCC1 has been shown to exist and possibly function as a homodimer. However, a possible heterocomplex involving ABCC1 has been indicated. In this study, we performed an interactive proteomics study to examine proteins that bind to and form heterocomplexes with ABCC1 using coimmunoprecipitation and tandem mass spectrometry (MS/MS) analyses. We found that ATP synthase α binds to ABCC1 in plasma membranes with a ratio of 2:1. The ATP synthase α binding site in ABCC1 is located in the linker domain at the carboxyl core of ABCC1, and phosphorylation of the linker domain at the protein kinase A site enhances ATP synthase α binding. The interaction between ABCC1 and ATP synthase α in a heterocomplex may indicate a novel function of ABCC1 in regulating extracellular ATP level and purinergic signaling cascade.

• Role of testosterone in stimulating seasonal changes in a potential avian chemosignal.

  Authors: Danielle J Whittaker, Helena A Soini, Nicole M Gerlach, Amanda L Posto, Milos V Novotny, Ellen D Ketterson

  Journal of chemical ecology. 12/2011; 37(12):1349-57.

  Songbird preen oil contains volatile and semivolatile compounds that may contain information about species, sex, individual identity, and season. We examined the relationship between testosterone (T)Songbird preen oil contains volatile and semivolatile compounds that may contain information about species, sex, individual identity, and season. We examined the relationship between testosterone (T) and the amounts of preen oil volatile and semivolatile compounds in wild and captive dark-eyed juncos (Junco hyemalis). In wild males and females, we observed an increase in volatile compound relative concentration early in the breeding season. This increase mirrored previously described seasonal elevation in T levels in wild males and females, suggesting a positive relationship between hormone levels and preen gland secretions, and a possible role for these secretions in signaling receptivity. In females, the greatest relative concentrations of most compounds were observed close to egg laying, a time when steroid hormones are high and also the only time that females respond to an injection of gonadotropin-releasing hormone with a short-term increase in T. In a study of captive juncos held on short days, we asked whether the seasonal increases observed in the wild could be induced with experimental elevation of T alone. We found that exogenous T stimulated the production of some volatile compounds in non-breeding individuals of both sexes. However, of the 15 compounds known to increase during the breeding season, only four showed an increase in relative concentration in birds that received T implants. Our results suggest that testosterone levels likely interact with other seasonally induced physiological changes to affect volatile compound amounts in preen oil.

• Delineating diseases by IMS-MS profiling of serum N-linked glycans.

  Authors: Dragan Isailovic, Manolo D Plasencia, Maissa M Gaye, Sarah T Stokes, Ruwan T Kurulugama, Vitara Pungpapong, Min Zhang, Zuzana Kyselova, Radoslav Goldman, Yehia Mechref, Milos V Novotny, David E Clemmer

  Journal of proteome research. 12/2011; 11(2):576-85.

  Altered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders theAltered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders the development of specific glycomic approaches for assessing disease states. Here, we examine a combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements, with principal component analysis (PCA) for characterizing serum N-linked glycans from 81 individuals: 28 with cirrhosis of the liver, 25 with liver cancer, and 28 apparently healthy. Supervised PCA of combined ion-mobility profiles for several, to as many as 10 different mass-to-charge ratios for glycan ions, improves the delineation of diseased states. This extends an earlier study [J. Proteome Res.2008, 7, 1109-1117] of isomers associated with a single glycan (S(1)H(5)N(4)) in which PCA analysis of the IMS profiles appeared to differentiate the liver cancer group from the other samples. Although performed on a limited number of test subjects, the combination of IMS-MS for different combinations of ions and multivariate PCA analysis shows promise for characterizing disease states.

• Microchip electrophoresis of N-glycans on serpentine separation channels with asymmetrically tapered turns.

  Authors: Zexi Zhuang, Indranil Mitra, Ahmed Hussein, Milos V Novotny, Yehia Mechref, Stephen C Jacobson

  Electrophoresis. 01/2011; 32(2):246-53.

  We designed and fabricated microfluidic devices with serpentine separation channels and asymmetrically tapered turns, thus allowing high efficiency separations and minimizing band broadeningWe designed and fabricated microfluidic devices with serpentine separation channels and asymmetrically tapered turns, thus allowing high efficiency separations and minimizing band broadening associated with the "racetrack" effect. We evaluated the performance of these devices by measuring the variation in separation efficiency with separation length, electric field strength, taper ratio of the turns, and number of turns. N-Glycans derived from ribonuclease B and labeled with 8-aminopyrene-1,3,6-trisulfonic acid were electrophoretically separated on serpentine channels with separation lengths of 11, 18, 22, and 36 cm at electric field strengths from 750 to 1750 V/cm. Separations on the 36-cm channel produced plate numbers up to 940,000 with an analysis time under 3.1 min, whereas separations on the 22-cm channel had a shorter analysis time (less than 1.25 min), still with respectable efficiencies (up to 600,000 plates). Turn-induced dispersion was minimized with taper ratios 2 and 3, whereas having two or four 180° turns along with the separation length did not impact the overall efficiency. The developed device was used to analyze native and desialylated N-glycans derived from the blood serum of an ovarian cancer patient and a disease-free individual. Separation efficiencies similar to that achieved with the model glycans from ribonuclease B were attained for these biological samples.

• Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement.

  Authors: Gaetano Vattemi, Yehia Mechref, Matteo Marini, Paola Tonin, Pietro Minuz, Laura Grigoli, Valeria Guglielmi, Iveta Klouckova, Cristiano Chiamulera, Alessandra Meneguzzi, Marzia Di Chio, Vincenzo Tedesco, Laura Lovato, Maurizio Degan, Guido Arcaro, Alessandro Lechi, Milos V Novotny, Giuliano Tomelleri

  Molecular & cellular proteomics : MCP. 12/2010; 10(4):M110.002964.

  Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may beMitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress.

• Analysis of site-specific glycosylation of renal and hepatic γ-glutamyl transpeptidase from normal human tissue.

  Authors: Matthew B West, Zaneer M Segu, Christa L Feasley, Pilsoo Kang, Iveta Klouckova, Chenglong Li, Milos V Novotny, Christopher M West, Yehia Mechref, Marie H Hanigan

  The Journal of biological chemistry. 09/2010; 285(38):29511-24.

  The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by aThe cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.

• Effects of lead and mercury on the blood proteome of children.

  Authors: Robert E Birdsall, Michael P Kiley, Zaneer M Segu, Christopher D Palmer, Milan Madera, Brooks B Gump, James A MacKenzie, Patrick J Parsons, Yehia Mechref, Milos V Novotny, Kestutis G Bendinskas

  Journal of proteome research. 09/2010; 9(9):4443-53.

  Heavy metal exposure in children has been associated with a variety of physiological and neurological problems. The goal of this study was to utilize proteomics to enhance the understanding ofHeavy metal exposure in children has been associated with a variety of physiological and neurological problems. The goal of this study was to utilize proteomics to enhance the understanding of biochemical interactions responsible for the health problems related to lead and mercury exposure at concentrations well below CDC guidelines. Blood plasma and serum samples from 34 children were depleted of their most abundant proteins using antibody-based affinity columns and analyzed using two different methods, LC-MS/MS and 2-D electrophoresis coupled with MALDI-TOF/MS and tandem mass spectrometry. Apolipoprotein E demonstrated an inverse significant association with lead concentrations (average being one microgram/deciliter) as deduced from LC-MS/MS and 2-D electrophoresis and confirmed by Western blot analysis. This coincides with prior findings that Apolipoprotein E genotype moderates neurobehavioral effects in individuals exposed to lead. Fifteen other proteins were identified by LC-MS/MS as proteins of interest exhibiting expressional differences in the presence of environmental lead and mercury.

• Analysis of volatile organic compounds in human saliva by a static sorptive extraction method and gas chromatography-mass spectrometry.

  Authors: Helena A Soini, Iveta Klouckova, Donald Wiesler, Elisabeth Oberzaucher, Karl Grammer, Sarah J Dixon, Yun Xu, Richard G Brereton, Dustin J Penn, Milos V Novotny

  Journal of chemical ecology. 09/2010; 36(9):1035-42.

  Human saliva not only helps control oral health (with anti-microbial proteins), but it may also play a role in chemical communication. As is the case with other mammalian species, human salivaHuman saliva not only helps control oral health (with anti-microbial proteins), but it may also play a role in chemical communication. As is the case with other mammalian species, human saliva contains peptides, proteins, and numerous volatile organic compounds (VOCs). A high-throughput analytical method is described for profiling a large number of saliva samples to screen the profiles of VOCs. Saliva samples were collected in a non-stimulated fashion. The method utilized static stir bar extraction followed by gas chromatography-mass spectrometry (GC-MS). The method provided excellent reproducibility for a wide range of salivary compounds, including alcohols, aldehydes, ketones, carboxylic acids, esters, amines, amides, lactones, and hydrocarbons. Furthermore, substantial overlap of salivary VOCs and the previously reported skin VOCs in the same subject group was found in this study by using pattern recognition analyses. Sensitivity, precision, and reproducibility of the method suggest that this technique has potential in physiological, metabolomic, pharmacokinetic, forensic, and toxicological studies of small organic compounds where a large number of human saliva samples are involved.

• Assigning N-glycosylation sites of glycoproteins using LC/MSMS in conjunction with endo-M/exoglycosidase mixture.

  Authors: Zaneer M Segu, Ahmed Hussein, Milos V Novotny, Yehia Mechref

  Journal of proteome research. 07/2010; 9(7):3598-607.

  The assignment of protein glycosylation sites and their microheterogeneities are of biological importance, yet such characterization is still considered to be analytically very challenging. SeveralThe assignment of protein glycosylation sites and their microheterogeneities are of biological importance, yet such characterization is still considered to be analytically very challenging. Several approaches have been recently developed to improve the characterization of glycosylation sites of proteins, including lectin and HILIC enrichment-based methods coupled to mass spectrometry. However, unequivocal assignment of protein glycosylation remains to be a daunting task, prompting continuous demands for the development of sensitive and cutting-edge analytical approaches. beta-N-Acetylglucosaminidase (endo-beta-GlcNAc-ases, Endo-M) is an endoglycosidase capable of hydrolyzing N,N'-diacetylchitobiose moiety in N-linked oligosaccharides bound to the asparagine amino acid residue in various glycoproteins. An attractive feature of this enzyme is its ability to cleave the N,N'-diacetylchitobiose moiety while leaving an N-acetylglucosamine residue bound to the protein. This enzyme is also known to be inactive in the presence of core fucose residue linked to the reducing-end N-acetylglucosamine residue (GlcNAc). Here, we describe an approach capitalizing on these features of Endo-M to (a) determine the glycosylation sites of proteins and the occupancy of these sites, and (b) determine the attachment sites of fucose residue containing N-glycans. The latter is important because of its biological implications. Tryptically digested glycoproteins, which were subjected to Endo-M treatment, were analyzed by LC-MS/MS. Systematic evaluation of the activity of Endo-M toward different glycan structures indicated a dependence of enzyme activity on the complexity of the glycan structures. Efficient release of N-glycans using Endo-M is only achieved through the inclusion of a battery of exoglycosidases to reduce the complexity of the attached glycans and subsequently prompt an effective enzymatic release. Upon Endo-M/exoglycosidase treatment of tryptically digested glycoproteins, glycosylated sites retain GlcNAc residue. The resulting peptides with GlcNAc residues attached to the glycosylation sites are easily assigned through LC-MS/MS analysis and subsequent database searching of the generated tandem MS of such entities. Comparing the LC-MS/MS results of the tryptic digest of glycoproteins treated with PNGase F and Endo-M/exoglycosidases allowed the assignment of core fucose residues to N-glycan reducing-ends. The detection of glycosylation sites only in the tryptic digest of PNGase F treated samples suggested core fucosylation of the attached N-glycans to such sites. This strategy was initially validated using model glycoproteins. It also proved to be useful in determining the glycosylation sites of blood serum glycoproteins.

• Chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated N-linked glycans: a potential methodology for cancer-biomarker discovery.

  Authors: William R Alley, Milan Madera, Yehia Mechref, Milos V Novotny

  Analytical chemistry. 06/2010; 82(12):5095-106.

  The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases.The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Quantification of glycomic-profile changes is becoming increasingly important in the search for disease biomarkers. Here, we report a highly reproducible combination of a glycomic sample preparation/solid-phase derivatization of glycoprotein-derived N-linked glycans with their subsequent microchip-based separation and mass-spectrometric (MS) measurements. Following our previously described reductive beta-elimination for O-linked glycans with ammonia-borane complex to reduce N-linked structures, the N-linked alditol structures are effectively methylated in dimethylformamide medium to avoid artefacts in MS measurements. Reversed-phase microfluidic liquid chromatography (LC) of methylated N-linked oligosaccharide alditols resolved some closely related structures into regular retention increments, aiding in their structural assignments. Optimized LC gradients, together with nanospray MS, have been applied here in the quantitative measurements of N-linked glycans in blood serum, distinguishing breast cancer patients from control individuals.

• A quantitative investigation of fucosylated serum glycoproteins with application to esophageal adenocarcinoma.

  Authors: Benjamin Mann, Milan Madera, Iveta Klouckova, Yehia Mechref, Lacey E Dobrolecki, Robert J Hickey, Zane T Hammoud, Milos V Novotny

  Electrophoresis. 06/2010; 31(11):1833-41.

  Although glycoproteomic studies provide unique opportunities for cancer research, it has been necessary to develop specific methods for analysis of oncologically interesting glycoproteins. WeAlthough glycoproteomic studies provide unique opportunities for cancer research, it has been necessary to develop specific methods for analysis of oncologically interesting glycoproteins. We describe a general, multimethodological approach for quantitative glycoproteomic analysis of fucosylated glycoproteins in human blood serum. A total of 136 putative fucosylated glycoproteins were identified with very high confidence in three clinically relevant sample pools (N=5 for each), with a mean CV of 3.1% observed for replicate analyses. Two samples were collected from subjects diagnosed with esophagus disease states, high-grade dysplasia plus esophageal adenocarcinoma, while the third sample was representative of a disease-free condition. Some glycoproteins, observed to be significantly upregulated in esophageal adenocarcinoma, i.e. more than twofold higher than in the disease-free condition, are briefly discussed. Further investigation will be necessary to validate these findings; however, the method itself is demonstrated to be an effective tool for quantitative glycoproteomics of clinical samples.

• Comparative glycomic profiling in esophageal adenocarcinoma.

  Authors: Zane T Hammoud, Yehia Mechref, Ahmed Hussein, Slavka Bekesova, Min Zhang, Kenneth A Kesler, Milos V Novotny

  The Journal of thoracic and cardiovascular surgery. 05/2010; 139(5):1216-23.

  Aberrant glycosylation has been implicated in various types of cancers. Cancerous cells with altered glycosylation of their surface proteins shed such proteins into the circulating fluids. GlycomicAberrant glycosylation has been implicated in various types of cancers. Cancerous cells with altered glycosylation of their surface proteins shed such proteins into the circulating fluids. Glycomic profiling of such fluids shows the altered glycosylation. We performed glycomic profiling of serum from patients with no known disease, Barrett's without dysplasia, with high-grade dysplasia, and with esophageal adenocarcinoma in an attempt to delineate distinct differences in glycosylation among these groups. Serum samples from patients with Barrett's metaplasia (N = 5), high-grade dysplasia (N = 11), and esophageal adenocarcinoma (N = 50) were collected; samples from 18 healthy volunteers were used as control. Serum N-glycans were enzymatically released and then applied to both C18 Sep-Pak (Waters, Milford, MA) cartridges and activated charcoal cartridges. N-glycans were permethylated and then spotted directly onto a matrix-assisted laser desorption ionization plate. Mass spectra were acquired using the Applied Biosystems 4800 MALDI TOF/TOF Analyzer (Applied Biosystems Inc, Framingham, Mass). The obtained matrix-assisted laser desorption ionization-mass spectrometry data were processed using DataExplorer files (Applied Biosystems Inc) listing m/z values and intensities. The intensities of 98 glycans were significantly different among the 3 groups; 26 of these corresponded to known glycan structures. Pairwise comparisons showed that 8 glycans were significantly different in all 3 pairwise comparisons. We demonstrated that comparative glycomic profiling of esophageal adenocarcinoma reveals a subset of glycans that can be selected as candidate biomarkers. These markers can differentiate normal from high-grade dysplasia, normal from esophageal adenocarcinoma, and high-grade dysplasia from esophageal adenocarcinoma. Further validation will be necessary to determine the clinical utility of these glycan biomarkers.

• Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

  Authors: Yoshinao Wada, Anne Dell, Stuart M Haslam, Bérangère Tissot, Kévin Canis, Parastoo Azadi, Malin Bäckström, Catherine E Costello, Gunnar C Hansson, Yoshiyuki Hiki [......] Milos V Novotny, Erina Ohno, Nicolle H Packer, Elizabeth Palaima, Matthew B Renfrow, Michiko Tajiri, Kristina A Thomsson, Hirokazu Yagi, Shin-Yi Yu, Naoyuki Taniguchi

  Molecular & cellular proteomics : MCP. 04/2010; 9(4):719-27.

  The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining theThe Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

• Mapping site-specific protein N-glycosylations through liquid chromatography/mass spectrometry and targeted tandem mass spectrometry.

  Authors: Yin Wu, Yehia Mechref, Iveta Klouckova, Anoop Mayampurath, Milos V Novotny, Haixu Tang

  Rapid communications in mass spectrometry : RCM. 03/2010; 24(7):965-72.

  Glycosylation is one of the most common posttranslational modifications (PTMs) of proteins, the characterization of which is commonly achieved through proteomic protocol, involving trypsin digestionGlycosylation is one of the most common posttranslational modifications (PTMs) of proteins, the characterization of which is commonly achieved through proteomic protocol, involving trypsin digestion followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, it is often not possible to characterize all glycopeptides in a complex sample because of the high complexity of glycoproteomic samples, and the relative lower abundances of glycopeptides in comparison to the unmodified peptides. We present here a targeted MS/MS analysis approach, which utilizes a previously developed computational tool, GlyPID, to guide multiple experiments, thus permitting a complete characterization of all N-glycosylation sites of glycoproteins present in a complex sample. We have tested our approach using model glycoproteins analyzed by high-resolution LTQ-FT MS. The results demonstrate a potential use of our method for a high-throughput characterization of complex mixtures of glycosylated proteins.

• Songbird chemosignals: volatile compounds in preen gland secretions vary among individuals, sexes, and populations.

  Authors: Danielle J Whittaker, Helena A Soini, Jonathan W Atwell, Craig Hollars, Milos V Novotny, Ellen D Ketterson

  Behavioral ecology : official journal of the International Society for Behavioral Ecology. 01/2010; 21(3):608-614.

  Chemical signaling has been documented in many animals, but its potential importance in avian species, particularly songbirds, has received far less attention. We tested whether volatile compounds inChemical signaling has been documented in many animals, but its potential importance in avian species, particularly songbirds, has received far less attention. We tested whether volatile compounds in the preen oil of a songbird (Junco hyemalis) contain reliable information about individual identity, sex, or population of origin by repeated sampling from captive male and female juncos originating from 2 recently diverged junco populations in southern California. One of the populations recently colonized an urban environment; the other resides in a species-typical montane environment. The birds were field-caught as juveniles, housed under identical conditions, and fed the same diet for 10 months prior to sampling. We used capillary gas chromatography-mass spectrometry to quantify the relative abundance of 19 volatile compounds previously shown to vary seasonally in this species. We found individual repeatability as well as significant sex and population differences in volatile profiles. The persistence of population differences in a common environment suggests that preen oil chemistry likely has a genetic basis and may thus evolve rapidly in response to environmental change. These finding suggest that songbird preen oil odors have the potential to function as chemosignals associated with mate recognition or reproductive isolation.

• Songbird chemosignals: volatile compounds in preen gland secretions vary among individuals, sexes, and populations

  Authors: Danielle J Whittaker, Helena A Soini, Jonathan W. Atwell, Craig Hollars, Milos V Novotny, Ellen D Ketterson

  Oxford University Press for the International Society for Behavioral Ecology, Behavioral Ecology. 01/2010; 21(3):608-614.

  Chemical signaling has been documented in many animals, but its potential importance in avian species, particularly songbirds, has received far less attention. We tested whether volatile compounds inChemical signaling has been documented in many animals, but its potential importance in avian species, particularly songbirds, has received far less attention. We tested whether volatile compounds in the preen oil of a songbird (Junco hyemalis) contain reliable information about individual identity, sex, or population of origin by repeated sampling from captive male and female juncos originating from 2 recently diverged junco populations in southern California. One of the populations recently colonized an urban environment; the other resides in a species-typical montane environment. The birds were field-caught as juveniles, housed under identical conditions, and fed the same diet for 10 months prior to sampling. We used capillary gas chromatography--mass spectrometry to quantify the relative abundance of 19 volatile compounds previously shown to vary seasonally in this species. We found individual repeatability as well as significant sex and population differences in volatile profiles. The persistence of population differences in a common environment suggests that preen oil chemistry likely has a genetic basis and may thus evolve rapidly in response to environmental change. These finding suggest that songbird preen oil odors have the potential to function as chemosignals associated with mate recognition or reproductive isolation. Copyright 2010, Oxford University Press.

• Enzymatic/Chemical Release of O-Glycans Allowing MS Analysis at High Sensitivity.

  Authors: John A Goetz, Milos V Novotny, Yehia Mechref

  Analytical chemistry. 10/2009;

  As the role of O-linked oligosaccharides have been demonstrated to be increasingly important in numerous medical conditions, it is imperative to develop new techniques allowing their analysis at highAs the role of O-linked oligosaccharides have been demonstrated to be increasingly important in numerous medical conditions, it is imperative to develop new techniques allowing their analysis at high sensitivity. While mass spectrometry (MS) provides adequate measurements of important O-linked oligosaccharides glycans and their profiles, the release from glycoproteins has not been sufficiently addressed for the needs of biomedical applications. This work describes a new strategy, involving the combination of a complete enzymatic degradation with a chemical release during the solid-phase permethylation of O-linked oligosaccharides. The analytical data implicate highly effective cleavage from the serine and threonine (but not arginine) residues, during permethylation. Tandem MS analyses confirmed these observations for model glycoproteins. Comparative measurements through isotopic labeling MS show this approach to be vastly superior over the previously used cleavage procedures.

• Sequential Enrichment of Sulfated Glycans by Strong Anion-Exchange Chromatography Prior to Mass Spectrometric Measurements.

  Authors: Ming Lei, Milos V Novotny, Yehia Mechref

  Journal of the American Society for Mass Spectrometry. 09/2009;

  Structural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ionStructural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ion mode. Here, we describe a microscale method for sequentially enriching sulfated glycans according to their degree of sulfation. This method is based on modifying the binding ability of strong anion-exchange material through the use of different sodium acetate concentrations, thus enabling fairly selective binding and a subsequent elution of different glycans according to their degree of sulfation. Before this enrichment, the negative charge on the sialic acid, which is commonly associated with such glycans, was eliminated through permethylation that is used to enhance the positive-ion mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) signal for all glycans. This enrichment approach minimizes competitive ionization between sulfated and neutral glycans, as well as that between sulfated species with a different degree of sulfation. The described method was initially optimized using sulfated oligosaccharide standards, while its potential has been verified for the sulfated N-glycans originated from the bovine thyroid-stimulating hormone (bTSH), a glycoprotein possessing mono- and disulfated N-glycans. This enhancement of the MALDI-MS signal facilitates analysis of some otherwise undetected components.

• Identification of N-glycan Serum Markers Associated with Hepatocellular Carcinoma from Mass Spectrometry Data.

  Authors: Zhiqun Tang, Rency S Varghese, Slavka Bekesova, Christopher Loffredo, Mohamed Abdel-Hamid, Zuzana Kyselova, Yehia Mechref, Milos V Novotny, Radoslav Goldman, Habtom W Ressom

  Journal of proteome research. 09/2009;

  Glycocylation represents the most complex and widespread post-translational modifications in human proteins. The variation of glycosylation is closely related to oncogenic transformation. Therefore,Glycocylation represents the most complex and widespread post-translational modifications in human proteins. The variation of glycosylation is closely related to oncogenic transformation. Therefore, profiling of glycans detached from proteins is a promising strategy to identify biomarkers for cancer detection. This study identified candidate glycan biomarkers associated with hepatocellular carcinoma by mass spectrometry. Specifically, mass spectrometry data were analyzed with a peak selection procedure which incorporates multiple random sampling strategies with recursive feature selection based on support vector machines. Ten peak sets were obtained from different combinations of samples. Seven peaks were shared by each of the ten peaksets, in which 7-12 peaks were selected, indicating 58%-100% of peaks were shared by the ten peaksets. SVM and hierarchical clustering method were used to evaluate the performance of the peak sets. The predictive performance of the seven peaks was further evaluated by using 19 MALDI-TOF spectra newly generated. Glycan structures for four glycans of the seven peaks were determined. Literature search indicated that these four glycan stuctures could be identified from the some cancer-related glycoproteins. The method of this study is significant in deriving consistent, accurate, and biological significant glycan marker candidates for hepatocellular carcinoma diagnosis.

• Quantitative Serum Glycomics of Esophageal Adenocarcinoma and Other Esophageal Disease Onsets (dagger).

  Authors: Yehia Mechref, Ahmed Hussein, Slavka Bekesova, Vitara Pungpapong, Min Zhang, Lacey E Dobrolecki, Robert J Hickey, Zane T Hammoud, Milos V Novotny

  Journal of proteome research. 06/2009;

  Aberrant glycosylation has been implicated in various types of cancers and changes in glycosylation may be associated with signaling pathways during malignant transformation. Glycomic profiling ofAberrant glycosylation has been implicated in various types of cancers and changes in glycosylation may be associated with signaling pathways during malignant transformation. Glycomic profiling of blood serum, in which cancer cell proteins or their fragments with altered glycosylation patterns are shed, could reveal the altered glycosylation. We performed glycomic profiling of serum from patients with no known disease (N = 18), patients with high grade dysplasia (HGD, N = 11) and Barrett's esophagus (N = 5), and patients with esophageal adenocarcinoma (EAC, N = 50) in an attempt to delineate distinct differences in glycosylation between these groups. The relative intensities of 98 features were significantly different among the disease onsets; 26 of these correspond to known glycan structures. The changes in the relative intensities of three of the known glycan structures predicted esophageal adenocarcinoma with 94% sensitivity and better than 60% specificity as determined by receiver operating characteristic (ROC) analysis. We have demonstrated that comparative glycomic profiling of EAC reveals a subset of glycans that can be selected as candidate biomarkers. These markers can differentiate disease-free from HGD, disease-free from EAC, and HGD from EAC. The clinical utility of these glycan biomarkers requires further validation.