Biomedical subjects

Milos V Novotny

Publications and source records attributed to Milos V Novotny.

At least 19 recordsLinked to original sources

PubMed2007

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

Carbohydrate Conformation↗

PubMed2007

Seasonal variation in volatile compound profiles of preen gland secretions of the dark-eyed junco (Junco hyemalis).

Quantitative stir bar sorptive extraction methodology, followed by gas chromatography-mass spectrometry (GC-MS) and element-specific atomic emission detection (AED) were utilized to analyze seasonal changes in volatile components of preen oil secretions in Junco hyemalis. Juncos were held in long days to simulate breeding conditions, or short days to simulate nonbreeding conditions. Linear alcohols (C(10)-C(18)) were the major volatile compounds found in preen oil, and in both sexes their levels were higher when birds were housed on long as opposed to short days. Methylketones were found at lower levels, but were enhanced in both sexes during long days. Levels of 2-tridecanone, 2-tetradecanone, and 2-pentadecanone were also greater on long days, but only in males. Among carboxylic acids (C(12), C(14), and C(16)), linear but not branched acids showed some differences between the breeding and nonbreeding conditions, although the individual variation for acidic compounds was large. Qualitatively, more sulfur-containing compounds were found in males than females during the breeding season. Functionally, the large increase in linear alcohols in male and female preen oil during the breeding season may be an indication of altered lipid biosynthesis, which might signal reproductive readiness. Linear alcohols might also facilitate junco odor blending with plant volatiles in the habitat to distract mammalian predators. Some of the volatile compounds from preen oil, including linear alcohols, were also found on the wing feather surface, along with additional compounds that could have been of either metabolic or environmental origin.

Animals↗

PubMed2007

Improved collision-induced dissociation analysis of peptides by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry through 3-sulfobenzoic acid succinimidyl ester labeling.

The sulfonation reagent, a succinimidyl ester of 3-sulfobenzoic acid, has been synthesized for effective peptide sequencing. It is capable of incorporating an additional mobile proton into the peptide backbone, thus, facilitating efficient collision-induced dissociation. This reagent is easily and inexpensively prepared in short time. Tandem mass spectra of the guanidinated and reagent-sulfonated peptides consist mainly of the y-ion series with higher intensities than those observed for solely guanidinated peptides. These enhanced tandem MS attributes significantly improved MASCOT total-ion scores, thus, allowing more confident peptide sequencing. This derivatization was also very effective for the analysis of tryptic digest of human blood serum proteins separated by two-dimensional gel electrophoresis. When used in LC-MALDI/MS/MS format, this type of derivatization does not adversely affect chromatographic efficiencies.

Amino Acid Sequence↗

PubMed2006

Chemical identification of MHC-influenced volatile compounds in mouse urine. I: Quantitative Proportions of Major Chemosignals.

The genes of the major histocompatibility complex (MHC) are highly polymorphic loci that encode cell surface proteins, class I and II molecules. They present peptide antigens to T cells and thereby control immunological self/nonself recognition. Increasing evidence indicates that MHC genes also influence odor and mating preferences; however, it is unclear how. Here we report the results of chemical analyses of male mouse urinary odors collected from a variety of mouse strains, including MHC-congenics, recombinants, mutants, and transgenics (i.e., beta2 microglobulin "knockouts," which lack class I expression, and transporters associated with antigen processing (TAP) knock-outs). After the identification of volatile odor components by gas chromatography/mass spectrometry, the odor profiles of urine samples were analyzed quantitatively by using stir bar sorptive extraction and gas chromatography/atomic emission detection. Results showed that MHC genes influenced the amounts of testosterone-mediated pheromones, sulfur-containing compounds, and several carbonyl metabolites. This is the first report to quantitatively link known mouse pheromones to classical, antigen-binding MHC loci. Surprisingly, these compounds were not influenced by TAP genes, even though these loci are MHC-linked and play a role in peptide presentation. Whereas identification of MHC-determined odorants does not reveal their metabolic origin, some constituents were also present in blood serum, and their levels were not altered by antibiotics.

Animals↗

PubMed2006

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker.

Two isoforms of proliferating cell nuclear antigen (PCNA) have been observed in breast cancer cells. Commercially available antibodies to PCNA recognize both isoforms and, therefore, cannot differentiate between the PCNA isoforms in malignant and nonmalignant breast epithelial cells and tissues. We have developed a unique antibody that specifically detects a PCNA isoform (caPCNA) associated with breast cancer epithelial cells grown in culture and breast-tumor tissues. Immunostaining studies using this antibody suggest that the caPCNA isoform may be useful as a marker of breast cancer and that the caPCNA-specific antibody could potentially serve as a highly effective detector of malignancy. We also report here that the caPCNA isoform functions in breast cancer-cell DNA replication and interacts with DNA polymerase delta. Our studies indicate that the caPCNA isoform may be a previously uncharacterized detector of breast cancer.

Antibodies↗

PubMed2006

In situ surface sampling of biological objects and preconcentration of their volatiles for chromatographic analysis.

This report describes a rolling stir bar sampling procedure for volatile organic compounds (VOCs) present on various biological surfaces. In combination with thermal desorption/gas chromatography/mass spectrometry, this analytical technique was initially tested for quantitative profiling of human skin VOCs. It is also applicable to additional hydrophobic surfaces such as agricultural products, plant materials, and bird feathers. Use of embedded internal standards provides highly reproducible and quantitative results for a wide variety of sampled trace components. The samples of collected human skin VOCs and standards were found stable under cool storage conditions for at least 14 days, making this approach suitable for field biological and agricultural studies. Additionally, this methodology appears to have potential for forensic and toxicological investigations, as suggested through the analyses of VOC profiles of the human thumb prints recovered from a nonbiological smooth surface.

Animals↗

PubMed2006

High-sensitivity profiling of glycoproteins from human blood serum through multiple-lectin affinity chromatography and liquid chromatography/tandem mass spectrometry.

We report here the use of high-performance lectin affinity enrichment of glycoproteins at microscale levels using a series of silica-bound lectins. The potential of this approach is being demonstrated for the glycoprotein enrichment from microliter volumes of human blood serum. Individual injections of sample to the affinity microcolumns packed with four lectin materials with different glycan specificities (Con A, SNA-I, UEA-I, PHA-L), followed by off-line reversed-phase pre-fractionation and nano-LC/MS/MS, permitted identification of 108 proteins in the lectin-bound fractions spanning a concentration dynamic range of 7-10 orders of magnitude. In contrast, multi-lectin microcolumn affinity chromatography, an alternative enrichment approach allowed identification of only 67 proteins. An attractive feature of high-performance lectin affinity chromatography at microscale levels is the substantial reduction of sample losses that are commonly experienced with extensive sample preparation needed for larger sample volumes.

Chromatography, Affinity↗

PubMed2006

A computational approach toward label-free protein quantification using predicted peptide detectability.

We propose here a new concept of peptide detectability which could be an important factor in explaining the relationship between a protein's quantity and the peptides identified from it in a high-throughput proteomics experiment. We define peptide detectability as the probability of observing a peptide in a standard sample analyzed by a standard proteomics routine and argue that it is an intrinsic property of the peptide sequence and neighboring regions in the parent protein. To test this hypothesis we first used publicly available data and data from our own synthetic samples in which quantities of model proteins were controlled. We then applied machine learning approaches to demonstrate that peptide detectability can be predicted from its sequence and the neighboring regions in the parent protein with satisfactory accuracy. The utility of this approach for protein quantification is demonstrated by peptides with higher detectability generally being identified at lower concentrations over those with lower detectability in the synthetic protein mixtures. These results establish a direct link between protein concentration and peptide detectability. We show that for each protein there exists a level of peptide detectability above which peptides are detected and below which peptides are not detected in an experiment. We call this level the minimum acceptable detectability for identified peptides (MDIP) which can be calibrated to predict protein concentration. Triplicate analysis of a biological sample showed that these MDIP values are consistent among the three data sets.

Algorithms↗

PubMed2006

Miniaturized separation techniques in glycomic investigations.

High-sensitivity glycomic analyses are becoming of a great interest in modern biomedical and clinical research, as well as in the development of recombinant protein products. The evolution of separation techniques for glycomic analysis at high sensitivity is highlighted in this review. These methodologies include capillary liquid chromatography, capillary electrophoresis (CE) and capillary electrochromatography (CEC). The potential of such methodologies in glycomic analysis is demonstrated for model glycoproteins as well as total glycomes derived from biological samples.

Chromatography, Liquid↗

PubMed2006

Determination of salsolinol and related catecholamines through on-line preconcentration and liquid chromatography/atmospheric pressure photoionization mass spectrometry.

A new analytical approach has been developed for simultaneous measurements of endogenous salsolinol and major catecholamines in brain tissue of experimental animals. This procedure involves a combination of on-line phenyl boronate affinity preconcentration and microcolumn liquid chromatography, followed by mass spectrometry equipped with an atmospheric pressure photoionization (APPI) source. Flow conditions of the APPI source were optimized for detection sensitivity while different dopants were evaluated. The on-line preconcentration was found essential for the sensitivity requirements of salsolinol measurements in the brain tissue from alcohol-preferring rats subjected to different levels of alcohol exposure.

Animals↗

PubMed2006

Chemotaxis assays of mouse sperm on microfluidic devices.

Sperm chemotaxis is an area of significant interest to scientists involved in reproductive science. Understanding how and when sperm cells are attracted to the egg could have profound effects on reproduction and contraception. In an effort to systematically study this problem, we have fabricated and evaluated a microfluidic device to measure sperm chemotaxis. The device was designed with a flow-through configuration using a spatially and temporally stable chemical gradient. Mouse sperm cells were introduced into the chemotaxis chamber between confluent flows of mouse ovary extract and buffer. The sperm experiencing chemotaxis swam toward the extract and were counted relative to those that swam toward the buffer. The ovary extracts were diluted from 10(2) to 10(7) times, and each extract dilution was screened for chemotaxis. Four out of six ovaries showed a strong chemotactic response at extract dilutions of 10(-3) to 10(-5). This device provided a convenient, disposable platform on which to conduct chemotaxis assays, and the flow-through design overcomes difficulties associated with distinguishing chemotaxis from trapping.

Animals↗

PubMed2006

Changes in liver protein abundance in inbred alcohol-preferring rats due to chronic alcohol exposure, as measured through a proteomics approach.

This study compares the total liver proteome of inbred alcohol-preferring line (iP) rats exposed to alcohol with iP rats without alcohol experience. Rat liver proteins were extracted using a three-step procedure. Each of the three solutions solubilizes a different set of proteins. The extracted proteins were separated by 2-DE. Scanned gels of two sample groups, alcohol-exposed iP and alcohol-naïve iP, were compared, revealing many protein spots with significantly higher or lower densities. These spots were cut from the gel, destained, and subjected to trypsin digestion and subsequent identification by LC-MS/MS. Twenty-four individual rats, 12 alcohol-naïve, and 12 alcohol-exposed, were used in this study. Two groups, each containing six naïve and six exposed animals, were created for statistical comparison. For the first group, 64 spots were observed to have statistically significant intensity differences upon alcohol exposure across all three extracts while 118 such spots were found in the second group. There were 113 unique proteins in both groups together. The majority of these proteins were enzymes. Significant changes are observed for three major metabolic pathways: glycolysis, gluconeogenesis, and fatty acid beta-oxidation. In addition, enzymes involved in protein synthesis and antioxidant activity show significant changes in abundance in response to alcohol exposure.

Alcoholism↗

PubMed2006

The discovery of labile methyl esters on proliferating cell nuclear antigen by MS/MS.

The post-translational modification of proliferating cell nuclear antigen (PCNA) has been implicated in modulating its function for over 20 years. With multiple interacting partners, PCNA is involved in processes ranging from DNA replication and repair to cell cycle control and apoptosis. The ability of PCNA to distinguish between specific binding partners in different tasks is currently of intense interest, and several post-translational modifications have been reported to modulate its function. Unfortunately, these reports have produced contradictory information on the type(s) of modification present on the molecule. Here we report a detailed structural analysis of a single acidic PCNA isoform, cancer-specific polyferating nuclear anitgen (csPCNA), isolated from breast cancer cells by 2D-PAGE and LC-MS/MS. With this approach we fully characterized the csPCNA isoform and confidently identified a single post-translational modification, methyl esterification. Interestingly, the methyl esters consistently localized to 15 specific glutamic and aspartic acid residues of csPCNA. The methyl esterification of csPCNA represents a novel type of post-translational modification in mammalian cells that could ultimately hold the key towards unlocking its diverse functions.

Amino Acid Sequence↗

PubMed2006

Differentiating structural isomers of sialylated glycans by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry.

Using model acidic glycans, we demonstrate the benefits of permethylation for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) tandem mass spectrometry. With both the linear and branched structures, extensive cross-ring fragmentation product ions were generated, yielding valuable information on sugar linkages. Elimination of the negative charges commonly associated with sialylated structures through permethylation allowed their structural analysis in the positive ion mode. Extensive A- and X-type ions were observed for the linear structures, and slightly weaker signals for the branched sialylated structures. The diagnostic cross-ring fragments, permitting a distinction between alpha2-3 and alpha2-6 linkages of the sialic acid residues, were seen in abundance. Importantly, the cross-ring fragmentation with the branched structures provides adequate information to assign sialic acid residues, with a specific linkage, to a particular antenna.

Glycosides↗

PubMed2006

Semiautomated high-sensitivity profiling of human blood serum glycoproteins through lectin preconcentration and multidimensional chromatography/tandem mass spectrometry.

We describe an effective analytical approach to identify trace glycoproteins in a small volume of human serum. The system is based on automatable affinity enrichment through silica-based lectin microcolumns and a further separation of the retained glycoproteins on a reversed-phase liquid chromatography with superficially porous packing, operating at high temperature. The fractionated sample is further directed into a 96-well plate for trypsinization and LC-MS/MS analysis. Using a major-component depleted blood serum (16 microg total protein), we were able to identify 271 glycoproteins through this analytical system.

Chromatography, Affinity↗

PubMed2005

Combining lectin microcolumns with high-resolution separation techniques for enrichment of glycoproteins and glycopeptides.

Silica-based lectin microcolumns are described in this study together with the chemical procedures necessary for their preparation. The analytical merits of Canavalia ensiformis and Sambucus nigra lectins, [immobilized on activated macroporous silica], such as binding capacity, trapping reproducibility, and substrate selectivity, have been evaluated using model glycoproteins. The described microcolumns are applicable to high-pressure analytical schemes utilizing microvalving procedures, washing steps, and quantitative desorption for LC/MS analysis. The described analytical systems are amenable to the applications aiming at fractionation of complex glycopeptide mixtures and determination of the sites of glycosylation.

Amino Acid Sequence↗

PubMed2005

Modulation of differentiation-related gene 1 expression by cell cycle blocker mimosine, revealed by proteomic analysis.

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

Amino Acid Sequence↗

PubMed2005

Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach.

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multimethodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgG(kappa))). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.

Amino Acid Sequence↗