Complexity of Chemical Emissions Increases Concurrently with Sexual Maturity in Heliconius Butterflies.

Pheromone communication is widespread among animals. Since it is often involved in mate choice, pheromone production is often tightly controlled. Although male sex pheromones (MSPs) and anti-aphrodisiacs have been studied in some Heliconius butterfly species, little is known about the factors affecting their production and release in these long-lived butterflies. Here, we investigate the effect of post-eclosion age on chemical blends from pheromone-emitting tissues in Heliconius atthis and Heliconius charithonia, exhibiting respectively free-mating and pupal-mating strategies that are hypothesised to differently affect the timing of their pheromone emissions. We focus on two different tissues: the wing androconia, responsible for MSPs used in courtship, and the genital tip, the production site for anti-aphrodisiac pheromones that affect post-mating behaviour. Gas chromatography-mass spectrometric analysis of tissue extracts from virgin males and females of both species from day 0 to 8 post-eclosion demonstrates the following. Some ubiquitous fatty acid precursors are already detectable at day 0. The complexity of the chemical blends increases with age regardless of tissue or sex. No obvious difference in the time course of blend production was evident between the two species, but female tissues in H. charithonia were more affected by age than in H. atthis. We suggest that compounds unique to male androconia and genitals and whose amount increases with age are potential candidates for future investigation into their roles as pheromones. While this analysis revealed some of the complexity in Heliconius chemical ecology, the effects of other factors, such as the time of day, remain unknown.

Exploitation of an ancestral pheromone biosynthetic pathway contributes to diversification in Heliconius butterflies.

During courtship, male butterflies of many species produce androconial secretions containing male sex pheromones (MSPs) that communicate species identity and affect female choice. MSPs are thus likely candidates as reproductive barriers, yet their role in speciation remains poorly studied. Although Heliconius butterflies are a model system in speciation, their MSPs have not been investigated from a macroevolutionary perspective. We use GC/MS to characterize male androconial secretions in 33 of the 69 species in the Heliconiini tribe. We found these blends to be species-specific, consistent with a role in reproductive isolation. We detected a burst in blend diversification rate at the most speciose genus, Heliconius; a consequence of Heliconius and Eueides species using a fatty acid (FA) metabolic pathway to unlock more complex blends than basal Heliconiini species, whose secretions are dominated by plant-like metabolites. A comparison of 10 sister species pairs demonstrates a striking positive correlation between blend dissimilarity and range overlap, consistent with character displacement or reinforcement in sympatry. These results demonstrate for the first time that MSP diversification can promote reproductive isolation across this group of butterflies, showcasing how implementation of an ancestral trait, the co-option of the FA metabolic pathway for pheromone production, can facilitate rapid speciation.

Surface Shave: Revealing the Apical-Restricted Uroglycome.

This study aimed to investigate the highly differentiated urothelial apical surface glycome. The functions of the mammalian urothelium, lining the majority of the urinary tract and providing a barrier against toxins in urine, are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification, and complex assembly to regulate the formation of multiple differentiated cell layers. Protein glycosylation, a poorly studied aspect of urothelial differentiation, contributes to the apical glycome and is implicated in the development of urothelial diseases. To enable surface glycome characterization, we developed a method to collect tissue apical surface N- and O-glycans. A simple, novel device using basic laboratory supplies was developed for enzymatic shaving of the luminal bladder urothelial surface, with subsequent release and mass spectrometric analysis of apical surface O- and N-glycans, the first normal mammalian urothelial N-glycome to be defined. Trypsinization of superficial glycoproteins was tracked using immunolabeling of the apically expressed uroplakin 3a protein to optimize enzymatic release, without compromising the integrity of the superficial urothelial layer. The approach developed for releasing apical tissue surface glycans allowed for comparison with the N-glycome of the total porcine bladder urothelial cells and thus identification of apical surface glycans as candidates implicated in the urothelial barrier function. Data are available in MassIve: MSV000087851.

Metabolomic Approaches to Studying the Response to Drought Stress in Corn (Zea mays) Cobs.

Metabolomics is a technique that allows for the evaluation of the entire extractable chemical profile of a plant, for example, using high-resolution mass spectrometry (HRMS) and can be used to evaluate plant stress responses, such as those due to drought. Metabolomic analysis is dependent upon the efficiency of the extraction protocol. Currently, there are two common extraction procedures widely used in metabolomic experiments, those that extract from plant tissue processed in liquid nitrogen or extraction from lyophilised plant tissues. Here, we evaluated the two using non-targeted metabolomics to show that lyophilisation can stabilise the maize (Zea mays) extractable metabolome, increasing throughput and efficiency of extraction as compared to the more traditional processing in liquid nitrogen. Then, we applied the lyophilisation approach to explore the effect of drought upon the maize metabolome in a non-targeted HRMS metabolomics approach. Metabolomics revealed differences in the mature maize metabolome having undergone three drought conditions imposed at two critical development stages (three-leaf stage and grain-fill stage); moreover, this difference was observed across two tissue types (kernel and inner cob/pith). It was shown that under ideal conditions, the biochemical make-up of the tissue types is different. However, under stress conditions, the stress response dominates the metabolic profile. Drought-related metabolites known from other plant systems have been identified and metabolomics has revealed potential novel drought-stress indicators in our maize system.

Pinus pinaster Early Hormonal Defence Responses to Pinewood Nematode (Bursaphelenchus xylophilus) Infection.

The pinewood nematode (PWN) is the causal agent of pine wilt disease, a pathology that affects conifer forests, mainly Pinus spp. PWN infection can induce the expression of phytohormone-related genes; however, changes at the early phytohormone level have not yet been explored. Phytohormones are low-abundance metabolites, and thus, difficult to quantify. Moreover, most methodologies focus mainly on Arabidopsis or crop species. This work aimed to validate a fast (run time 6.6 min) liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) analytical method to quantify 14 phytohormones in Pinus pinaster stem tissues. This method was further applied to evaluate, for the first time, early phytohormone changes in susceptible and resistant phenotypes of P. pinaster 24, 48 and 72 h after inoculation (HAI) with PWN. A significant increase in salicylic acid (SA, 48 and 72 HAI) and jasmonic acid methyl ester (JA-ME, 72 HAI) was observed in susceptible phenotypes. Results indicate that the higher susceptibility of P. pinaster to PWN infection might result from an inefficient trigger of hypersensitive responses, with the involvement of JA and SA pathways. This work provides an important update in forest research, and adds to the current knowledge of Pinus spp. defence responses to PWN infection.

Pyrene Tags for the Detection of Carbohydrates by Label-Assisted Laser Desorption/Ionisation Mass Spectrometry*.

Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) is widely used for the analysis of biomolecules. Label-assisted laser desorption/ionisation mass spectrometry (LALDI-MS) is a matrix-free variant of MALDI-MS, in which only analytes covalently attached to a laser desorption/ionisation (LDI) enhancer are detected. LALDI-MS has shown promise in overcoming the limitations of MALDI-MS in terms of sample preparation and MS analysis. In this work, we have developed a series of pyrene-based LDI reagents (LALDI tags) that can be used for labelling and LALDI-MS analysis of reducing carbohydrates from complex (biological) samples without the need for additional chemical derivatisation or purification. We have systematically explored the suitability of four pyrene-based LDI enhancers and three aldehyde-reactive handles, optimised sample preparation, and demonstrated the use of LALDI tags for the detection of lactose. We have also exemplified the potential of LALDI tags for labelling carbohydrates in biological samples by direct detection of lactose in cow's milk. These results demonstrate that LALDI-MS is a promising technique for the analysis of reducing carbohydrates in biological samples, and pave the way for the development of LALDI-MS for glycomics and diagnostics.

Five structural genes required for ceramide synthesis in Caulobacter and for bacterial survival.

Sphingolipids are essential and common membrane components in eukaryotic organisms, participating in many important cellular functions. Only a few bacteria are thought to harbour sphingolipids in their membranes, among them the well-studied α-proteobacterium Caulobacter crescentus, a model organism for asymmetric cell division and cellular differentiation. Here, we report that C. crescentus wild type produces several molecular species of dihydroceramides, which are not produced in a mutant lacking the structural gene for serine palmitoyltransferase (spt). Whereas growth of a spt-deficient mutant and wild type are indistinguishable during the exponential phase of growth, survival of the spt-deficient mutant is much reduced, in comparison with wild type, during stationary phase of growth, especially at elevated temperatures. The structural gene for spt is located within a genomic cluster, comprising another 16 genes and which, like spt, are important for fitness of C. crescentus. Mutants deficient in genes linked to spt by high cofitness were unable to produce dihydroceramide or to survive in stationary phase of growth at elevated temperatures. At least five structural genes are required for dihydroceramide biosynthesis in C. crescentus and sphingolipid biosynthesis is needed for survival of this bacterium and the integrity of its outer membrane.

The N-Glycosylation Processing Potential of the Mammalian Golgi Apparatus.

Heterogeneity is an inherent feature of the glycosylation process. Mammalian cells often produce a variety of glycan structures on separate molecules of the same protein, known as glycoforms. This heterogeneity is not random but is controlled by the organization of the glycosylation machinery in the Golgi cisternae. In this work, we use a computational model of the N-glycosylation process to probe how the organization of the glycosylation machinery into different cisternae drives N-glycan biosynthesis toward differing degrees of heterogeneity. Using this model, we demonstrate the N-glycosylation potential and limits of the mammalian Golgi apparatus, for example how the number of cisternae limits the goal of achieving near homogeneity for N-glycans. The production of specific glycoforms guided by this computational study could pave the way for "glycoform engineering," which will find uses in the functional investigation of glycans, the modulation of glycan-mediated physiological functions, and in biotechnology.

Modeling Glycan Processing Reveals Golgi-Enzyme Homeostasis upon Trafficking Defects and Cellular Differentiation.

The decoration of proteins by carbohydrates is essential for eukaryotic life yet heterogeneous due to a lack of biosynthetic templates. This complex carbohydrate mixture-the glycan profile-is generated in the compartmentalized Golgi, in which level and localization of glycosylation enzymes are key determinants. Here, we develop and validate a computational model for glycan biosynthesis to probe how the biosynthetic machinery creates different glycan profiles. We combined stochastic modeling with Bayesian fitting that enables rigorous comparison to experimental data despite starting with uncertain initial parameters. This is an important development in the field of glycan modeling, which revealed biological insights about the glycosylation machinery in altered cellular states. We experimentally validated changes in N-linked glycan-modifying enzymes in cells with perturbed intra-Golgi-enzyme sorting and the predicted glycan-branching activity during osteogenesis. Our model can provide detailed information on altered biosynthetic paths, with potential for advancing treatments for glycosylation-related diseases and glyco-engineering of cells.

Ionisation bias undermines the use of matrix-assisted laser desorption/ionisation for estimating peptide deamidation: Synthetic peptide studies demonstrate electrospray ionisation gives more reliable response ratios.

RATIONALE: Although mass spectrometry (MS) is routinely used to determine deamination in peptide mixtures, the effects of the choice of ionisation source have not yet been investigated. In particular, matrix-assisted laser desorption/ionisation (MALDI) has become a popular tool with which to measure levels of glutamine deamidation in ancient proteins. Here we use model synthetic peptides to rigorously compare MALDI and electrospray ionisation (ESI). METHODS: We used two synthetic peptides, with glutamine (Q) in one substituted for glutamic acid (E) in the other, to investigate the suitability of MALDI and ESI sources for the assessment of deamidation in peptides using MS. We also compared measurements of the same Q- and E-containing peptide mixtures using two different mass analysers (time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR)). RESULTS: When standard mixtures of the Q- and E-containing peptides were analysed using MALDI, under-representation of the E-containing peptide was observed. This observation was consistent between analyses carried out using either TOF or FT-ICR-MS. When the same mixtures were analysed using ESI FT-ICR-MS, no ionisation bias was observed. CONCLUSIONS: MALDI may not be a suitable ionisation method for the determination of deamidation in peptide mixtures. However, ESI was successfully used to determine the ratio in known mixtures of Q- and E-containing peptides. These preliminary observations warrant further investigation into ionisation bias when measuring deamidation in other peptide sequences.

A subcompatible rhizobium strain reveals infection duality in Lotus.

Lotus species develop infection threads to guide rhizobia into nodule cells. However, there is evidence that some species have a genetic repertoire to allow other modes of infection. By conducting confocal and electron microscopy, quantification of marker gene expression, and phenotypic analysis of transgenic roots infected with mutant rhizobia, we elucidated the infection mechanism used by Rhizobium leguminosarum Norway to colonize Lotus burttii. Rhizobium leguminosarum Norway induces a distinct host transcriptional response compared with Mesorhizobium loti. It infects L. burttii utilizing an epidermal and transcellular infection thread-independent mechanism at high frequency. The entry into plant cells occurs directly from the apoplast and is primarily mediated by 'peg'-like structures, the formation of which is dependent on the production of Nod factor by the rhizobia. These results demonstrate that Lotus species can exhibit duality in their infection mechanisms depending on the rhizobial strain that they encounter. This is especially relevant in the context of interactions in the rhizosphere where legumes do not encounter single strains, but complex rhizobial communities. Additionally, our findings support a perception mechanism at the nodule cell entry interface, reinforcing the idea that there are successive checkpoints during rhizobial infection.

Distinctive phytohormonal and metabolic profiles of Arabidopsis thaliana and Eutrema salsugineum under similar soil drying.

MAIN CONCLUSIONS: Arabidopsis and Eutrema show similar stomatal sensitivity to drying soil. In Arabidopsis, larger metabolic adjustments than in Eutrema occurred, with considerable differences in the phytohormonal responses of the two species. Although plants respond to soil drying via a series of concurrent physiological and molecular events, drought tolerance differs greatly within the plant kingdom. While Eutrema salsugineum (formerly Thellungiella salsuginea) is regarded as more stress tolerant than its close relative Arabidopsis thaliana, their responses to soil water deficit have not previously been directly compared. To ensure a similar rate of soil drying for the two species, daily soil water depletion was controlled to 5-10% of the soil water content. While partial stomatal closure occurred earlier in Arabidopsis (Day 4) than Eutrema (from Day 6 onwards), thereafter both species showed similar stomatal sensitivity to drying soil. However, both targeted and untargeted metabolite analysis revealed greater response to drought in Arabidopsis than Eutrema. Early peaks in foliar phytohormone concentrations and different sugar profiles between species were accompanied by opposing patterns in the bioactive cytokinin profiles. Untargeted analysis showed greater metabolic adjustment in Arabidopsis with more statistically significant changes in both early and severe drought stress. The distinct metabolic responses of each species during early drought, which occurred prior to leaf water status declining, seemed independent of later stomatal closure in response to drought. The two species also showed distinct water usage, with earlier reduction in water consumption in Eutrema (Day 3) than Arabidopsis (Day 6), likely reflecting temporal differences in growth responses. We propose Arabidopsis as a promising model to evaluate the mechanisms responsible for stress-induced growth inhibition under the mild/moderate soil drying that crop plants are typically exposed to.

GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation.

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.

Detection of opium alkaloids in a Cypriot base-ring juglet.

A method has been developed for extracting poppy alkaloids from oily matrices, specifically lipid residues associated with archaeological ceramics. The protocol has been applied to fresh and artificially aged poppyseed oil and to residue from a Late Bronze Age Cypriot juglet in the collections of the British Museum. The juglet is of a type that has been linked with ancient trade in opium due to its poppy-head shape and wide distribution; it is a rare example of an intact vessel with contents sealed inside. Bulk analysis of the residue by GC-EI-MS and pyGC-EI-MS indicated a degraded plant oil and possible presence of papaverine. Analysis of the alkaloid extracts by HPLC-ESI-MS using both triple quadrupole and FTICR mass spectrometers detected the five primary opium alkaloids in fresh poppyseed oil and papaverine in most of the aged samples. Papaverine and thebaine were detected in the juglet residue, providing the first rigorous chemical evidence to support a link between this vessel type and opium, or at least poppies. The association of opium with oil raises new questions about the ancient purpose of the commodities within these vessels, and the low levels (ng g-1) of opiates detected in this unusually well-preserved residue shed doubt on the scope for their detection in more fragmentary ceramic remains (potsherds). Papaverine was found to exhibit challenging carryover behaviour in all the analytical methods used in this study. The phenomenon has not been reported before and should be considered in future analyses of this analyte in all application areas.

Application of prioritization approaches to optimize environmental monitoring and testing of pharmaceuticals.

Pharmaceuticals are ubiquitous in the natural environment with concentrations expected to rise as human population increases. Environmental risk assessments are available for a small portion of pharmaceuticals in use, raising concerns over the potential risks posed by other drugs that have little or no data. With >1900 active pharmaceutical ingredients in use, it would be a major task to test all of the compounds with little or no data. Desk-based prioritization studies provide a potential solution by identifying those substances that are likely to pose the greatest risk to the environment and which, therefore, need to be considered a priority for further study. The aim of this review was to (1) provide an overview of different prioritization exercises performed for pharmaceuticals in the environment and the results obtained; and (2) propose a new holistic risk-based prioritization framework for drugs in the environment. The suggested models to underpin this framework are discussed in terms of validity and applicability. The availability of data required to run the models was assessed and data gaps identified. The implementation of this framework may harmonize pharmaceutical prioritization efforts and ensure that, in the future, experimental resources are focused on molecules, endpoints, and environmental compartments that are biologically relevant.

Polyamines are required for normal growth in Sinorhizobium meliloti.

Polyamines (PAs) are ubiquitous polycations derived from basic l-amino acids whose physiological roles are still being defined. Their biosynthesis and functions in nitrogen-fixing rhizobia such as Sinorhizobium meliloti have not been extensively investigated. Thin layer chromatographic and mass spectrometric analyses showed that S. meliloti Rm8530 produces the PAs, putrescine (Put), spermidine (Spd) and homospermidine (HSpd), in their free forms and norspermidine (NSpd) in a form bound to macromolecules. The S. meliloti genome encodes two putative ornithine decarboxylases (ODC) for Put synthesis. Activity assays with the purified enzymes showed that ODC2 (SMc02983) decarboxylates both ornithine and lysine. ODC1 (SMa0680) decarboxylates only ornithine. An odc1 mutant was similar to the wild-type in ODC activity, PA production and growth. In comparison to the wild-type, an odc2 mutant had 45 % as much ODC activity and its growth rates were reduced by 42, 14 and 44 % under non-stress, salt stress or acid stress conditions, respectively. The odc2 mutant produced only trace levels of Put, Spd and HSpd. Wild-type phenotypes were restored when the mutant was grown in cultures supplemented with 1 mM Put or Spd or when the odc2 gene was introduced in trans. odc2 gene expression was increased under acid stress and reduced under salt stress and with exogenous Put or Spd. An odc1 odc2 double mutant had phenotypes similar to the odc2 mutant. These results indicate that ODC2 is the major enzyme for Put synthesis in S. meliloti and that PAs are required for normal growth in vitro.

Temporal and spatial variation in pharmaceutical concentrations in an urban river system.

Many studies have quantified pharmaceuticals in the environment, few however, have incorporated detailed temporal and spatial variability due to associated costs in terms of time and materials. Here, we target 33 physico-chemically diverse pharmaceuticals in a spatiotemporal exposure study into the occurrence of pharmaceuticals in the wastewater system and the Rivers Ouse and Foss (two diverse river systems) in the city of York, UK. Removal rates in two of the WWTPs sampled (a conventional activated sludge (CAS) and trickling filter plant) ranged from not eliminated (carbamazepine) to >99% (paracetamol). Data comparisons indicate that pharmaceutical exposures in river systems are highly variable regionally, in part due to variability in prescribing practices, hydrology, wastewater management, and urbanisation and that select annual median pharmaceutical concentrations observed in this study were higher than those previously observed in the European Union and Asia thus far. Significant spatial variability was found between all sites in both river systems, while seasonal variability was significant for 86% and 50% of compounds in the River Foss and Ouse, respectively. Seasonal variations in flow, in-stream attenuation, usage and septic effluent releases are suspected drivers behind some of the observed temporal exposure variability. When the data were used to evaluate a simple environmental exposure model for pharmaceuticals, mean ratios of predicted environmental concentrations (PECs), obtained using the model, to measured environmental concentrations (MECs) were 0.51 and 0.04 for the River Foss and River Ouse, respectively. Such PEC/MEC ratios indicate that the model underestimates actual concentrations in both river systems, but to a much greater extent in the larger River Ouse.

Trehalose During Two Stress Responses in Acanthamoeba: Differentiation Between Encystation and Pseudocyst Formation.

The non-reducing disaccharide trehalose can serve as a protectant against a range of environmental stressors, such as heat, cold, or dehydration, in both prokaryotes and eukaryotes, with the exception of vertebrates. Here, we analyzed trehalose metabolism in the facultatively parasitic organism Acanthamoeba castellanii, known to respond to unfavorable external conditions by forming two resistant stages: a cyst, produced in the case of chronic stress, and a pseudocyst, formed in reaction to acute stress. The possible role of trehalose in the resistant stages was investigated using a combination of bioinformatic, molecular biological and biochemical approaches. Genes for enzymes from a widespread trehalose-6-synthase-trehalose-6-phosphate phosphatase (TPS-TPP) pathway and a prokaryotic trehalose synthase (TreS) pathway were identified. The expression patterns of the genes during encystation and pseudocyst formation were analyzed and correlated with the time course of cellular trehalose content determined mass spectrometrically. The data clearly demonstrate fundamental differences between encystation and pseudocyst formation at the level of cellular metabolism.

Are exposure predictions, used for the prioritization of pharmaceuticals in the environment, fit for purpose?

Prioritization methodologies are often used for identifying those pharmaceuticals that pose the greatest risk to the natural environment and to focus laboratory testing or environmental monitoring toward pharmaceuticals of greatest concern. Risk-based prioritization approaches, employing models to derive exposure concentrations, are commonly used, but the reliability of these models is unclear. The present study evaluated the accuracy of exposure models commonly used for pharmaceutical prioritization. Targeted monitoring was conducted for 95 pharmaceuticals in the Rivers Foss and Ouse in the City of York (UK). Predicted environmental concentration (PEC) ranges were estimated based on localized prescription, hydrological data, reported metabolism, and wastewater treatment plant (WWTP) removal rates, and were compared with measured environmental concentrations (MECs). For the River Foss, PECs, obtained using highest metabolism and lowest WWTP removal, were similar to MECs. In contrast, this trend was not observed for the River Ouse, possibly because of pharmaceutical inputs unaccounted for by our modeling. Pharmaceuticals were ranked by risk based on either MECs or PECs. With 2 exceptions (dextromethorphan and diphenhydramine), risk ranking based on both MECs and PECs produced similar results in the River Foss. Overall, these findings indicate that PECs may well be appropriate for prioritization of pharmaceuticals in the environment when robust and local data on the system of interest are available and reflective of most source inputs. Environ Toxicol Chem 2017;36:2823-2832. © 2017 SETAC.

One Filter, One Sample, and the N- and O-Glyco(proteo)me: Toward a System to Study Disorders of Protein Glycosylation.

A method has been developed for release/isolation of O-glycans from glycoproteins in whole cell lysates for mass spectrometric analysis. Cells are lysed in SDS, which is then exchanged for urea and ammonium bicarbonate in a centrifugal filter, before treating with NH4OH to release O-glycans. Following centrifugation, O-glycans are recovered in the filtrate. Sonication achieves O-glycan release in 1 h. Combining the established protocol for filter-aided N-glycan separation, here optimized for enhanced PNGase F efficiency, with the developed O-glycan release method allows analysis of both N- and O-glycans from one sample, in the same filter unit, from 0.5 to 1 million cells. The method is compatible with subsequent analysis of the residual protein by liquid chromatography-mass spectrometry (LC-MS) after glycan release. The medium throughput approach is amenable to analysis of biological replicates, offering a simple way to assess the often subtle changes to glycan profiles accompanying differentiation and disease progression, in a statistically robust way.