Glycomic profiles derived from human blood sera of 10 healthy males were compared to those from 24 prostate cancer patients. The profiles were acquired using MALDI-MS of permethylated N-glycans released from 10-μl sample aliquots. Quantitative permethylation was attained using solid-phase permethylation. Principal component analysis of the glycomic profiles revealed significant differences among the two sets, allowing their distinct clustering. The first principal component distinguished the 24 prostate cancer patients from the healthy individuals. It was determined that fucosylation of glycan structures is generally higher in cancer samples (ANOVA test p-value of 0.0006). Although more than 50 N-glycan structures were determined, twelve glycan structures, of which six were fucosylated, were significantly different between the two sample sets. Significant differences were confirmed through two independent statistical tests (ANOVA and ROC analyses). Ten of these structures had a significantly higher relative intensities in the case of the cancer samples, while the other two were less abundant in the cancer samples. All 12 structures were statistically significant, as suggested by their very low ANOVA scores (< 0.001) and ROC analysis, with area under the curve values close to 1 or zero. Accordingly, these structures can be considered as cancer–specific glycans and potential prostate cancer biomarkers. Therefore, serum glycomic profiling appears worthy of further investigation to define its role in cancer early detection and prognostication.
prostate cancer; glycomics; human blood serum; biomarkers; MALDI-TOF; mass spectrometry
Among the most important proteins involved in the disease and healing processes are the immunoglobulins (Igs). Although many of the Igs have been studied through proteomics, aside from IgG, immunoglobulin carbohydrates have not been extensively characterized in different states of health. It seems valuable to develop techniques that permit us to understand changes in the structures and abundances of Ig glycans in the context of disease onset and progression. We have devised a strategy for characterization of the glycans for the Ig classes other than IgG (i.e. A, D, E, and M) that contain kappa light chains, while using only a few microliters of biological material. First, we designed a microcolumn containing the recombinant Protein L that was immobilized on macroporous silica particles. A similarly designed Protein G microcolumn was utilized to first perform an on-line depletion of the IgG from the sample, human blood serum, and thereby facilitate enrichment of the other Igs. While only 3 μL of serum were used in these analyses, we were able to recover a significantly-enriched fraction of non-IgG immunoglobulins. The enrichment properties of the Protein L column were characterized using a highly sensitive label-free quantitative proteomics LC-MS/MS approach, and the glycomic profiles of enriched immunoglobulins were measured by MALDI-TOF-MS. As a proof-of-principle, a comparative study was conducted using blood serum from a small group of lung cancer patients and a group of age-matched cancer-free individuals to demonstrate that the method is suitable for investigation of glycosylation changes in disease. The results were in agreement with a glycomic investigation of whole blood serum from a much larger lung cancer cohort.
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 (IgGk)). 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.
Glycoproteins; Glycosylation sites; N-Glycans; Recombinant monoclonal antibody
High-sensitivity glycoprotein analyses are of particular interest in modern biomedical and clinical research, as well as in the development of recombinant protein products. The evolution of new hyphenated methodologies in high-sensitivity glycoprotein analysis is highlighted in this thematic review. These methodologies include, in particular, capillary LC/MALDI/TOF/TOF MS in conjunction with online permethylation platform, and silica-based lectin microcolumns interfaced to MS. The potential of these methodologies in glycomic and glycoproteomic analysis is demonstrated for model glycoproteins as well as total glycomes and glycoproteomes derived from biological samples. Additionally, the applications of CE-MS, CEC, and nanoLC with graphitized carbon in the areas of glycomics and glycoproteomics are described.
Glycomics; Glycoproteomics; LC/MALDI/TOF/TOF MS; LC/MS; Lectin; Oligosaccharides; Online permethylation; Review; Tandem mass spectrometry
Among of the most urgent needs of the glycobiology community is to generate libraries of pure carbohydrate standards. While many oligosaccharides have recently been synthesized, some glycans of biomedical importance are still missing in existing collections, or are available in only limited amounts. To address this need, we demonstrate the use of the relatively unexplored technique of recycling high-performance liquid chromatography (R-HPLC) to isolate and purify glycoconjugates from several natural sources. We were able to routinely achieve purities greater than 98%. In several cases, we were able to obtain isomerically pure substances, particularly for glycans with different positional isomerism. These purified substances can then be used in different analytical applications, for example, as standards for mass spectrometry (MS) and capillary-based separations. Moreover, using a bifunctional aromatic amine, the same derivatization agent can be used to enable UV detection of oligosaccharides during their purification and link the isolated molecules to functionalized surfaces and potentially create glycan arrays.
recycling HPLC; hydrophilic-interaction chromatography; glycans; carbohydrates; glycan arrays; 4-(2-aminoethyl)aniline
Ovarian cancer is the fifth leading cause of cancer-related mortalities for women in the United States and the most lethal gynecological cancer. Aberrant glycosylation has been linked to several human diseases, including ovarian cancer, and accurate measurement of changes in glycosylation may provide relevant diagnostic and prognostic information. In this work, we used microchip electrophoresis coupled with laser-induced fluorescence detection to determine quantitative differences among the N-glycan profiles of control individuals and late-stage recurrent ovarian cancer patients prior to and after an experimental drug treatment that combined docetaxel and imatinib mesylate. N-Glycans were enzymatically released from 5-μL aliquots of serum samples, labeled with the anionic fluorescent tag, 8-aminopyrene-1,3,6-trisulfonic acid, and analyzed on microfluidic devices. A 22-cm long separation channel, operated at 1250 V/cm, generated analysis times less than 100 s, separation efficiencies up to 8 × 105 plates (3.6 × 106 plates/m), and migration time reproducibilities better than 0.1% relative standard deviation after peak alignment. Principal component analysis (PCA) and analysis of variance (ANOVA) tests showed significant differences between the control and both pre- and post-treatment cancer samples and subtle differences between the pre- and post-treatment cancer samples. Area-under-the curve (AUC) values from receiver operating characteristics (ROC) tests were used to evaluate the diagnostic merit of N-glycan peaks, and specific N-glycan peaks used in combination provided AUCs > 0.90 (highly accurate test) when the control and pre-treatment cancer samples and control and post-treatment samples were compared.
microfluidics; microchip electrophoresis; N-glycans; glycan profiling; disease monitoring; ovarian cancer; glycomics; multiple biomarkers
The great complexity of glycosylated biomolecules necessitates a set of powerful analytical methodologies to reveal functionally important structural features. Mass spectrometry (MS), with its different ionization techniques, mass analyzers, and detection strategies, has become the most important analytical method in glycomic and glycoproteomic investigations. In combination with MS, microscale separations (based on capillary chromatography and electrophoresis) and carbohydrate microchemistry, we feature here conceptually important applications of the recent years. This review focuses on methodological advances pertaining to disease biomarker research, immunology, developmental biology, and measurements of importance to biopharmaceuticals. High-sensitivity determinations and sample enrichment/preconcentration are particularly emphasized in glycomic and glycoproteomic profiling.
The field of glycomics has recently advanced in response to the urgent need for structural characterization and quantification of complex carbohydrates in biologically and medically important applications. The recent success of analytical glycobiology at high sensitivity reflects numerous advances in biomolecular mass spectrometry and its instrumentation, capillary and microchip separation techniques, and microchemical manipulations of carbohydrate reactivity. The multimethodological approach appears to be necessary to gain an in-depth understanding of very complex glycomes in different biological systems.
biomolecular mass spectrometry; carbohydrates; permethylation; porous graphitic carbon; sialic acids; capillary electrophoresis
A new, mechanically stable silica microparticle with macro-sized internal pores (1.6-μm particles with 100-nm pores) has been developed for chromatography. The particles are characterized by an extensive network of interconnected macropores with a high intraparticle void volume as observed by transmission electron microscopy (TEM). They are synthesized by an aerosol assembly technique called ultrasonic spray pyrolysis (USP). The particles have a high surface area for a macroporous material, ~200 m2/g, making them suitable for large biomolecular separations. To demonstrate their potential for bioseparations, they have been functionalized with lectins for affinity enrichment of glycoproteins. The material was derivatized with two lectins, Concanavalin A (Con A) and Aleuria aurantia lectin (AAL), and binding properties were tested with standard glycoproteins. The columns exhibited excellent binding capacities for microaffinity enrichment—Con A was able to bind 75 μg of a standard glycoprotein in a 50 × 1-mm column. Following initial tests, the lectin microcolumns were utilized for enrichment of glycoproteins from 1-μL volumes of blood serum samples, which was performed in triplicate for each lectin. The enriched serum fractions were subjected to side-by-side glycomic and glycoproteomic profiling analyses with mass spectrometry to show that the new particles offer excellent sensitivity for microscale analyses of precious biological sample materials. The unique combination of the macroporous architecture and small particle diameter suggests the material may have advantages for conventional modes of chromatographic separation of macromolecules in an ultrahigh-pressure liquid chromatography (UHPLC) format.
This review summarizes the analytical advances made during the last several years in the structural and quantitative determinations of glycoproteins in complex biological mixtures. The main analytical techniques used in the fields of glycomics and glycoproteomics involve different modes of mass spectrometry and their combinations with capillary separation methods such as microcolumn liquid chromatography and capillary electrophoresis. The needs for high-sensitivity measurements have been emphasized in the oligosaccharide profiling used in the field of biomarker discovery through MALDI mass spectrometry. High-sensitivity profiling of both glycans and glycopeptides from biological fluids and tissue extracts has been aided significantly through lectin preconcentration and the uses of affinity chromatography.
biomolecular mass spectrometry; glycoproteomics; glycomics; affinity chromatography; lectin enrichment; permethylation; sialic acid linkage analysis; capillary electrophoresis
Agouti is a secreted neuropeptide that acts as an endogenous antagonist of melanocortin receptors. Mice and rats lacking agouti (called non-agouti) have dark fur due to a disinhibition of melanocortin signaling and pigment deposition in the hair follicle. Non-agouti animals have also been reported to exhibit altered behavior, despite no evidence for the expression of agouti outside the skin. Here we confirm that non-agouti mice show altered social behavior and uncover expression of agouti in the preputial gland, a sebaceous organ in the urinary tract that secretes molecules involved in social behavior. Non-agouti mice had enlarged preputial glands and altered levels of putative preputial pheromones and surgical removal of the gland reversed the behavioral phenotype. These findings demonstrate the existence of an autologous, out-of-skin pathway for the modulation of social behavior.
social behavior; agouti; melanocortin receptors; preputial glands; volatile organic compounds (VOCs); sorptive stir bar extraction (SBSE)
Glycosylation is a key post-translational protein modification which appears important in malignant transformation and tumor metastasis. Abnormal glycosylation of different proteins can often be measured in the blood serum. In this study, we extend our serum-based structural investigations to samples provided by patients diagnosed with lung cancer, paying particular attention to the effects of smoking on the serum glycomic traces. Following a battery of glycomic tests, we find that several fucosylated tetra-antennary structures with varying degrees of sialylation are increased in their abundances in control samples provided by the former smokers, with further elevations in the lung cancer patients who were former smokers. Further detailed investigations demonstrated that the level of outer-arm fucosylation was elevated in the control samples of the former smokers and again in the lung cancer samples provided by the former smokers. This trend was particularly noticeable for the tri- and tetra-antennary structures. Different ratios of sialylation linkages were also observed that could be correlated with the different states of health and smoking status. Decreases in the abundance levels of isomers with two and three α2,3-linked sialic acids and an increased abundance of an isomer with two α2,6-linked sialic acids were noted for a fucosylated tri-sialylated tri-antennary glycan. These results demonstrate the long-term effects of smoking on glycomic profiles and that this factor needs to be considered in these and other serum-based analyses.
disease markers; glycomics; non-small cell lung carcinoma; permethylation; smoking
We 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 (RNase 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 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 RNase B were attained for these biological samples.
microchip electrophoresis; asymmetric turns; serpentine microchannels; N-glycans; ovarian cancer
Hepatocellular cancer is a serious human disease with an unfortunately low survival rate. It further poses a significant epidemic threat to our society through its viral vectors associated with cirrhosis conditions preceding the cancer. A search for biomarkers of these diseases enlists analytical glycobiology, in general, and quantitative biomolecular mass spectrometry (MS), in particular, as valuable approaches to cancer research. The recent advances in quantitative glycan permethylation prior to MALDI-MS oligosaccharide profiling has enabled us to compare the glycan quantitative proportions in the small serum samples of cancer and cirrhotic patients against control individuals. We were further able to fractionate the major serum proteins from the minor components and compare statistically their differential glycosylation, elucidating some causes of quantitatively unusual glycosylation events. Numerous glycan structures were tentatively identified and connected with the origin proteins, with a particular emphasis on sialylated and fucosylated glycans.
glycomics; hepatocellular carcinoma; permethylation; immunoaffinity fractionation; MALDI-TOF MS; glycans
Reproducible and efficient affinity enrichment is increasingly viewed as an essential step in many investigations leading to the discovery of new biomarkers. In this work, we have evaluated the repeatability of lectin enrichment of glycoproteins from human blood serum through both qualitative and quantitative proteomic approaches. In a comprehensive evaluation of lectin binding, we have performed 30 separate microscale lectin affinity chromatography experiments, followed by a conventional sample purification, and LC-MS/MS analysis of the enriched glycoproteins. Two lectin affinity matrixes, both with Con A lectin immobilized to the same solid support but differing in the amount of immobilized lectin, were investigated to characterize their binding properties. Both qualitative and quantitative data indicate acceptable repeatability and binding efficiency for the lectin materials received from two different commercial sources.
Glycoproteins; Lectin Affinity Chromatography; Quantitative Glycoproteomics; LC-MS/MS
Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip-based liquid chromatographic system. The capabilities of both activated graphitized carbon and C18 LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C18 chips. On the other hand, hydrophobic glycopeptides were better retained on C18 chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (< 1 pmol). The performance of both media was also appeared comparable when analyzing a 4-protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase.
Activated graphitized carbon chip; Glycopeptides; LC/MSMS
Glycosylated proteins play important roles in a broad spectrum of biochemical and biological processes, and prior reports have suggested that changes in protein glycosylation occur during cancer initiation and progression. Ovarian cancer (OC) is a fatal malignancy, most commonly diagnosed after the development of metastases. Therefore, early detection of OC is key to improving survival. To this end, specific changes of the serum glycome have been proposed as possible biomarkers for different types of cancers. In this study, we extend this concept to OC. To characterize differences in total N-glycan levels, serum samples provided by 20 healthy control women were compared to those acquired from patients diagnosed with late-stage recurrent OC who were enrolled in an experimental treatment trial prior to receiving therapy (N = 19) and one month later, prior to the second treatment cycle (N = 11). Additionally, analyses of the N-glycans associated with IgG and characterization of the relative abundance levels of core vs. outer-arm fucosylation were also performed. The N-linked glycomic profiles revealed increased abundances of tri- and tetra-branched structures with varying degrees of sialylation and fucosylation and an apparent decrease in the levels of “bisecting” glycans in OC samples compared to controls. Increased levels of a-galactosylated structures were observed on N-linked glycans derived from IgG, which were independent of the presence of fucose residues. Elevated levels of outer-arm fucosylation were also identified in the OC samples. These results allowed the control samples to be distinguished from the baseline ovarian cancer patients prior to receiving the experimental treatment. In some cases, the pre-treatment samples could be distinguished from the post-experimental treatment samples, as many of those patients showed a further progression of the disease.
ovarian cancer; permethylation; disease markers; fucosylation; IgG; glycomics; exoglycosidase digestion
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 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 ten 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 (S1H5N4) 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.
ion mobility; ESI mass spectrometry; glycans; cancer; principal component analysis
Adult neurogenesis in female mice is known to be enhanced by exposure to soiled bedding from males, although the identity of the relevant chemosignals has remained unknown. Here we show that the previously recognized male murine pheromones, the farnesenes and 2-sec-butyl-4,5-dihydrothiazole (SBT), strongly increase cell proliferation in the subventricular zone (SVZ) of adult female mice, but not younger female mice. In addition, we found that a unique female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. SBT stimulated cell proliferation in the SVZ of only adult females and not in young adult or pre- and post-puberty females. Our study suggests that pheromonal communication between males and females is enhancing reproductive success by controlling the estrous cycle and by promoting cell proliferation in a reciprocal manner.
pheromones; enhanced cell proliferation; subventricular zone; male; female; mice
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 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.
Proteomics; children; blood; cardiovascular; lead; mercury; apolipoprotein E; Pb; Hg; ApoE
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 β-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.
LC-MS; cancer; blood serum glycoproteins; glycans; biomarker; ammonia-borane complex; permethylation
A number of alterations to the normal glycomic profile have been previously described for a number of diseases and disorders, thus underscoring the medical importance of studying the glycans associated with proteins present in biological samples. An important alteration in cancer progression is an increased level of α2,6-sialylation, which aids in increasing the metastatic potential of tumor cells. Here we report a glycomic method that selectively amidates α2,6-linked sialic acids, while those that are α2,3-linked undergo spontaneous lactonization. Following subsequent permethylation, MALDI-TOF-MS analysis revealed that many sialylated glycans present on glycoproteins found in blood serum featured increased levels of α2,6 sialylation in breast cancer samples. Based on the altered ratios of α2,3-linked to α2,6-linked sialic acids, many of these glycans became diagnostically relevant when they did not act as such indicators when based on traditional glycomic profiling alone.
cancer; sialic acid; permethylation; amidation; lactonization
Whereas 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 coefficient of variation of 3.1% observed for replicate analyses. Two samples were collected from subjects diagnosed with esophagus disease states, high-grade dysplasia (HGD) plus esophageal adenocarcinoma (EAC), while the third sample was representative of a disease-free (DF) condition. Some glycoproteins, observed to be significantly upregulated in EAC, i.e. more than 2-fold higher than in the DF 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.
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 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.
birds; chemical communication; Junco hyemalis; olfaction; pheromones