Milos V. Novotny

Ultrasensitive pheromone detection by mammalian vomeronasal neurons.

The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones into electrical responses that regulate sexual, hormonal and reproductive function in mammals. The characteristics of pheromone signal detection by vomeronasal neurons remain unclear. Here we use a mouse VNO slice preparation to show that six putative pheromones evoke excitatory responses in single vomeronasal neurons, leading to action potential generation and elevated calcium entry. The detection threshold for some of these chemicals is remarkably low, near 10-11 M, placing these neurons among the most sensitive chemodetectors in mammals. Using confocal calcium imaging, we map the epithelial representation of the pheromones to show that each of the ligands activates a unique, nonoverlapping subset of vomeronasal neurons located in apical zones of the epithelium. These neurons show highly selective tuning properties and their tuning curves do not broaden with increasing concentrations of ligand, unlike those of receptor neurons in the main olfactory epithelium. These findings provide a basis for understanding chemical signals that regulate mammalian communication and sexual behaviour.

Individual and gender fingerprints in human body odour

Individuals are thought to have their own distinctive scent, analogous to a signature or fingerprint. To test this idea, we collected axillary sweat, urine and saliva from 197 adults from a village in the Austrian Alps, taking five sweat samples per subject over 10 weeks using a novel skin sampling device. We analysed samples using stir bar sorptive extraction in connection with thermal desorption gas chromatograph–mass spectrometry (GC–MS), and then we statistically analysed the chromatographic profiles using pattern recognition techniques. We found more volatile compounds in axillary sweat than in urine or saliva, and among these we found 373 peaks that were consistent over time (detected in four out of five samples per individual). Among these candidate compounds, we found individually distinct and reproducible GC–MS fingerprints, a reproducible difference between the sexes, and we identified the chemical structures of 44 individual and 12 gender-specific volatile compounds. These individual compounds provide candidates for major histocompatibility complex and other genetically determined odours. This is the first study on human axillary odour to sample a large number of subjects, and our findings are relevant to understanding the chemical nature of human odour, and efforts to design electronic sensors (e-nose) for biometric fingerprinting and disease diagnoses.

Increased protein backbone conformational entropy upon hydrophobic ligand binding.

For complexes between proteins and very small hydrophobic ligands, hydrophobic effects alone may be insufficient to outweigh the unfavorable entropic terms resulting from bimolecular association. NMR relaxation experiments indicate that the backbone flexibility of mouse major urinary protein increases upon binding the hydrophobic mouse pheromone 2-sec-butyl-4,5-dihydrothiazole. The associated increase in backbone conformational entropy of the protein appears to make a substantial contribution toward stabilization of the protein–pheromone complex. This term is likely comparable in magnitude to other important free energy contributions to binding and may represent a general mechanism to promote binding of very small ligands to macromolecules.

Chemistry of male dominance in the house mouse,Mus domesticus

Two terpenic constituents, E,E,-α-farnesene and E-β-farnesene, were found to be elevated in dominant male urine when compared to subordinate or control males. These two urinary compounds were absent in the bladder urine of males; however, they were the most prominent constituents of the preputial glands aliquots. The results of a two-choice preference test, conducted on ICR/Alb subordinate males, gave a strong indication that these two terpenic constituents introduced into the previously attractive stimulus significantly discouraged prolonged investigations by male mice. The compounds, whether present in the urine matrix or water, rendered the stimulus with a quality behaviorally similar to the urine of dominant males. It appears that they may be synonymous with the previously described aversion signal produced by dominant males. We suggest that these compounds may play a wide-ranging role in the territorial marking behavior of male mice.

Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein

Five structurally diverse small ligands, all binding to the major urinary protein (MUP) of the male house mouse, show individually puberty-accelerating pheromonal activity in the recipient females. A recombinant MUP (identical structurally to the natural protein) has shown no biological activity. While four of these ligands were previously implicated in oestrus synchronization (Whitten effect), the same chemosignals now appear responsible for both sexual maturation and cycling in adult females.

Performance evaluation of slurry-packed capillary columns for liquid chromatography

SummaryFused-silica capillary columns with 200 μm internal diameter were efficiently packed with both 3 μm and 5 μm spherical C18 bonded-phase particles. Optimum packing conditions were determined and column performance was then critically evaluated in terms of the reduced plate height as a function of both reduced velocity and solute capacity factor. Characteristic values for various column performance indices, such as the Knox separation impedance and the interparticle porosity, were also determined and found to be highly reproducible and similar to those of conventional columns. At optimum velocity, the column contribution to reduced plate height values appeared very close to the theoretical. The columns were found to tolerate samples of up to 5 μg with little loss of efficiency and were used in the high-resolution separation of coal-derived fluids.

High-sensitivity Analytical Approaches for the Structural Characterization of Glycoproteins

1.1. General Considerations Structural intricacies of carbohydrate molecules and their propensity to form varied linkages, substitutions, and branching patterns have fascinated many generations of chemists, as have the three-dimensional aspects of carbohydrate interactions with other biomolecules. The steadily increasing biochemical knowledge in this area has further added to the increasing importance of the field now referred to as “glycobiology” or, more generally, “glycoscience”. Yet, most of the emphasis over the last 50 years or so has been on two other classes of important biopolymers, namely nucleic acids and proteins. However, in the “post-genomic era”, complex carbohydrates can no longer be neglected, as it is becoming clear to many scientists that most mammalian proteins are glycosylated, and microbial systems and plants can have their own unique monosaccharide building blocks and special ways they can be interconnected and branched into unusual structures. Throughout evolution and the development of living organisms, glycoconjugates must have played major roles, no doubt due to their unusual biological selectivities, which, in turn, could well be due to the enormous information capacity of the “sugar code”.1,2 Throughout the 1980s, the multilateral importance of glycoconjugates in biology and medicine was recognized,3-6 albeit with an understanding that only new methodological approaches and systematic investigations would further define new vistas and provide intimate knowledge of how complex carbohydrates participate in all life processes. Today’s glycoscience is a multidisciplinary undertaking in which chemistry is expected to have an important role to describe the most complex structural aspects of sugars and their conjugates with other biological molecules. While the biological and biomedical relevance of studying glycosylation and sugar–protein and sugar–sugar interactions will undoubtedly be guided by advances in other respective fields (immunology, cancer research, parasitology, cell biology, and developmental biology, among others), the chemical disciplines’ two major tasks are to (a) isolate and structurally characterize biologically important glycoconjugates and (b) synthesize carbohydrate structures for biochemical investigations, enabling technologies and medical applications and providing new therapeutics. While the goals and directions of carbohydrate synthesis have been summarized elsewhere,7-11 the focus of our review has been on glycoanalytical chemistry. The synthetic and bioanalytical directions are not mutually exclusive, as new structural findings will undoubtedly provide further rationale for synthetic efforts and these, in turn, the availability of standards for structural verification. Since publication of the review on structural investigations of glycoconjugates at high sensitivity12 in these pages a decade ago, the field of analytical glycobiology has seen dramatic changes in its scope and depth. It is widely appreciated within the glycoscience community and increasingly by others that both new techniques and instrumentation and the established (albeit optimized) analytical methodologies have played very important roles in advancing the science of glycoconjugates to its current stage. Due to their different physical and chemical characteristics, the main classes of glycoconjugates, i.e. glycoproteins, glycolipids, polysaccharides, and proteoglycans with their highly charged constituents, glycosaminoglycans, demand somewhat specialized analytical and structural elucidation approaches. Our review will largely be focused on glycoproteins and their associated glycans, hoping that other scientists will describe the analytical aspects of the remaining glycoconjugate biomolecules elsewhere. The early advances in proteomics, the scientific area mostly preoccupied with identification and structural characterization of proteins, have led to diverse activities in protein post-transitional modifications (PTMs), which are often associated with important biological activities. Glycosylation of proteins is arguably the most widely spread and functionally most intriguing PTM in nature. It is already known that certain glycosylation patterns in proteins give rise to functional variance, with far-reaching consequences for health-disease issues, immunological disorders, toxicity effects, microbial invasion processes, etc. To investigate any of these highly important processes in sufficient molecular detail, analytical techniques capable of a high degree of structural elucidation and measurement sensitivity are currently needed. Within the plethora of new “-omics fields” (genomics, transcriptomics, lipidomics, metabolomics, etc.), the fields of glycoproteomics and glycomics have started to assume their respectable roles. Analytical glycobiology, representing both glycomics and glycoproteomics, now shares access to new measurement technologies that enable characterization and quantification of molecular processes in living organisms. Extensive glycomic and glycoproteomic data that can nowadays be generated with modern techniques and instrumentation are likely to enrich the “systems biology” approach.13-17 Both fields have started to contribute substantially to a better understanding of multicellular interactions in eukaryotic systems and important issues pertaining to human health and disease.18-23 Additionally, the long-held view that glycosylation is unimportant in prokaryotic systems is no longer defensible.24,25 Since our previous review12 in this journal, much progress has been achieved in terms of methodological developments toward better, more informative, and more sensitive measurements of glycoproteins and their glycan components. In addition, many conceptually important applications of new tools already point to the future needs for dealing with the enormous complexity of glycopeptides and oligosaccharide mixtures extracted from biological tissues and physiological fluids. The relatively recent interest of the pharmaceutical and biotech industries in recombinant glycoproteins, such as monoclonal antibodies, for treatment of cancer and other diseases,26-30 demands the use and further development of glycomic and glycoproteomic analytical procedures as well. Similarly to our previous report,12 the current review has been organized to discuss separately recent advances in glycoproteomics and glycomics, dealing first with the isolation and direct analysis of glycoproteins, followed by the description of advances in glycopeptide analysis and determination of the sites of glycosylation, and moving toward the analysis of complex glycan mixtures. Even more today than 10 years ago, mass spectrometry (MS) is the most prominent methodology in the arsenal of glycoprotein analysis tools. A number of new MS techniques, previously unexplored or insufficiently developed, are now at the center of attention of glycobiologists. At the sensitivity levels required by contemporary glycobiology, MS and tandem MS (MSn) techniques are currently the only means to provide reliable structural information. Carbohydrate derivatization (chemical modification of carbohydrates at microscale) uniquely enables certain MS measurements in terms of enhanced sensitivity and structural information. Due to the enormous “chemical space” for carbohydrate structural complexity,1,2 MS alone, no matter how sophisticated, is unlikely to provide all needed answers. However, in combinations with modern separation methodologies (different forms of chromatography and electrophoresis) that provide unique component resolution in time and space, MS detection and identification capabilities become enormously enriched. The past decade has seen substantial improvements in the chromatographic analysis of complex carbohydrates: (1) transition from the conventional-scale columns to capillary column dimensions, or even microchips, with the resulting gains in mass sensitivity of measurements; and (2) rapidly increasing use of stable and reliable hydrophilic column materials and graphitized carbon adsorbents. Further advances in capillary chromatographic separations pertain to effective resolution of very complex mixtures as well as the frequently needed separation of different isomers. Chromatographic advances of the recent years also relate to simple purifications of samples (analysis steps now often referred to as solid-phase extraction, or SPE) or the more sophisticated microcolumn lectin or affinity materials needed in group separations and preconcentration of certain glycoproteins for analysis. The past decade has also witnessed a rapid development of glycan array technologies, in which the surface-bound glycan structures (either synthesized or isolated from natural mixtures) are presented to glycan-binding proteins in biological samples.31-33 While these enabling technologies are novel and exciting, they will not be covered in this review, which primarily emphasizes techniques leading to structural elucidation of glycoproteins. Likewise, immunologically based measurements will not be discussed.

Breast cancer diagnosis and prognosis through quantitative measurements of serum glycan profiles.

BACKGROUND Glycosylated proteins play important roles in cell-to-cell interactions, immunosurveillance, and a variety of receptor-mediated and specific protein functions through a highly complex repertoire of glycan structures. Aberrant glycosylation has been implicated in cancer for many years. METHODS We performed specific MALDI mass spectrometry (MS)-based glycomic profile analyses of permethylated glycans in sera from breast cancer patients (12, stage I; 11, stage II; 9, stage III; and 50, stage IV) along with sera from 27 disease-free women. The serum glycoproteins were enzymatically deglycosylated, and the released glycans were purified and quantitatively permethylated before their MALDI-MS analyses. We applied various statistical analysis tools, including ANOVA and principal component analysis, to evaluate the MS profiles. RESULTS Two statistical procedures implicated several sialylated and fucosylated N-glycan structures as highly probable biomarkers. Quantitative changes according to a cancer stage resulted when we categorized the glycans according to molecular size, number of oligomer branches, and abundance of sugar residues. Increases in sialylation and fucosylation of glycan structures appeared to be indicative of cancer progression. Different statistical evaluations confirmed independently that changes in the relative intensities of 8 N-glycans are characteristic of breast cancer (P < 0.001), whereas other glycan structures might contribute additionally to distinctions in the statistically recognizable patterns (different stages). CONCLUSIONS MS-based N-glycomic profiling of serum-derived constituents appears promising as a highly sensitive and informative approach for staging the progression of cancer.

Urinary volatile constituents of the house mouse, Mus musculus, and their endocrine dependency

Mouse urine contains a great number of volatile constituents that may be used in chemical communication. Some of these volatiles, identified in this study by combined gas chromatography-mass spectrometry and gas chromatography-Fourier-transform infrared spectroscopy, appear unique to the mouse. Certain urinary volatiles exhibit strong dependence on the sex and endocrine status of the animals, as shown through castration, treatment with an antiandrogen, and hormone supplementation.

Selected applications of cyclodextrin selectors in capillary electrophoresis.

Through the use of alpha-, beta-, gamma- and heptakis(2,6-di-O-methyl)-beta-cyclodextrin as stereospecific selectors or electrolyte modifiers, both in capillary zone electrophoresis and isotachophoresis, selected model isomeric compounds (including optical isomers) were resolved. Soluble alkylhydroxyalkylcellulose derivatives were further added to the cyclodextrin-modified background electrolytes under study. Their presence was found to be essential, as demonstrated by improvements in both enantioselectivity and separation efficiency. The results obtained in both electrophoretic modes, under optimized conditions, are compared and discussed.