Characterization of Humulus lupulus glycosides with porous graphitic carbon and sequential high performance liquid chromatography quadrupole time-of-flight mass spectrometry and high performance liquid chromatography fractionation.

Monoterpenes contribute to the characteristic aroma of several hop varieties and may occur as nonvolatile glycosides. Upon hydrolysis, the volatile terpenes are released from the glycoside precursors. Little is known, however, about the glycoside composition of hops. Seven pentose-hexose monoterpene alcohol glycosides from dried Humulus lupulus L. cv. Citra cones were isolated using high performance liquid chromatography separation and fractionation on a reverse phase phenyl-hexyl column. Further evaluation of each isolated fraction through HPLC qTOF MS with porous graphitic carbon (PGC) showed that the seven isolated monoterpenyl glycoside fractions could be further resolved into 20 isomers. Isolation on phenyl-hexyl followed by separation on PGC was needed to distinguish each isomer present. Additionally, the hop cones were screened for potential aroma glycosides. Using the PGC column combined with a database of over 900 potential glycosides, the identification of 21 additional monoterpene-polyol, norisoprenoid, volatile phenol, and aliphatic alcohol glycosides is reported.

Changes in ethylene and sugar metabolism regulate flavonoid composition in climacteric and non-climacteric plums during postharvest storage.

Plums are rich in flavonoids, key contributors to fruit coloration and putative health benefits. We studied the impact of changes in ethylene and sugars in flavonoid metabolism-related pathways of the climacteric Santa Rosa and its non-climacteric mutant Sweet Miriam, throughout the postharvest period. Fruits were harvested at optimal maturity, subjected to ethylene treatments, and evaluated during storage. We examined transcript profiles of structural and regulatory genes of flavonoid-related pathways and their associated metabolites in skin and flesh, integrated with multivariate analyses of ethylene and sugar metabolism. Ethylene treatments were positively correlated with anthocyanin and negatively correlated with flavonol and flavan-3-ol metabolism. Sucrose and galactose were positively associated with anthocyanin concentration, while sorbitol, fructose, glucose and minor sugars were correlated with flavonol and flavan-3-ol metabolism. Our results support the notion that ethylene is playing key roles in shifting plum fruit flavonoid profiles, which are also associated with changes in fruit sugars.

Antibiotic standards stored as a mixture in water: methanol are unstable at various temperatures irrespective of pH and glass container silanization.

It is well-established that antibiotics stored individually at their optimal pH and in appropriate solvents are stable over time. However, limited information exists on the stability of antibiotics from multiple classes when prepared and stored as a mixture prior to multiresidue analysis by mass spectrometry. This study tested the stability of antibiotic standard mixtures from eight classes [amphenicols, tetracyclines, sulfonamides, quinolones, macrolides, ß-lactams, lincosamides and miscellaneous (i.e., trimethoprim)] in relation to the water:methanol ratio, presence of sodium hydroxide base (to solubilise quinolones), storage temperature, and container type including plain and silanized glass vials. Antibiotics were analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. Several antibiotics, mainly quinolones, tetracyclines and macrolides, were unstable when stored as mixtures for one week regardless of the water:methanol ratio, storage temperature (4, -20 or -80 °C) and presence/absence of sodium hydroxide. Silanization of glassware improved the storage stability of quinolones and macrolides but reduced the stability of tetracyclines and other antibiotics including florfenicol amine, penicillin G, erythromycin and sulfadiazine. Our results show that several antibiotics in water:methanol are unstable when stored as a mixture and suggest a limited advantage of using base or silanized glass vials for the preparation and storage of antibiotic standards mixed together. Freshly prepared antibiotic standard mixtures are recommended for multi-residue quantitation of antibiotics.Abbreviations AMOX: amoxicillin; AMP: ampicillin; AZ: azithromycin; CAP: chloramphenicol; CE: collision energy; CTC: chlortetracycline; CIP: ciprofloxacin; DOX: doxycycline; ENO: enoxacin; ENRO: enrofloxacin; ERYTH: erythromycin; FF: florfenicol; FFA: florfenicol amine; FLU: flumequine; HDPE: high-density polyethylene; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LIN: lincomycin; MRM: multiple reaction monitoring; NOR: norfloxacin; OFL-D3: ofloxacin-D3; OXO: oxolinic acid; OTC: oxytetracycline; PEN-G: penicillin G; PEN-V: penicillin V; ROX: roxithromycin; SDM: sulfadimethoxine; SDZ: sulfadiazine; SMX: sulfamethoxazole; SMZ-D4: sulfamethazine-D4; SSZ: sulfasalazine; TC: tetracycline; TAP: thiamphenicol; TILM: tilmicosin; TRIM: trimethoprim; TL: tolerance limit; VIRG: virginiamycin; UPLC-MS/MS: ultra-high pressure liquid chromatography-tandem mass spectrometry.

Characterization of Free and Bound Monoterpene Alcohols during Riesling Fermentation.

The isomeric nature of monoterpenyl glycosides makes unambiguous identification of intact glycosides difficult. As a result, it is challenging to relate the changes in free monoterpenol concentrations to the corresponding glycosides during wine fermentation and storage. In this study, we isolated and identified linalool, nerol, and geraniol monoterpenyl glycosides fromVitis viniferacv. Riesling grapes through fractionation followed by acid or enzyme hydrolysis. Changes in the composition of identified monoterpenyl glycosides and their respective free volatiles were then monitored during alcoholic fermentations of Riesling juice with four different yeast strains across two successive years. The relative concentrations of the volatiles were monitored by solid-phase microextraction gas chromatography mass spectrometry, while ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry was used for intact glycosides. Glycoside hydrolysis during fermentation could be related to relative concentrations of the corresponding free aglycones. However, other sources of free monoterpenols were also observed. Differences in glycoside hydrolysis among yeast strains and across years were observed and may be related to grape maturity and/or nutrient levels.

Impact of Dry Hopping on Beer Flavor Stability.

To investigate the chemical and sensorial impact of dry hopping time on typical pale ale, a standardized beer was produced and separated into ten vessels. Nine vessels were dry hopped, and one vessel remained un-hopped as a control. Impact of dry hopping contact time was investigated over 96 h. Polyphenols and iso-α-acid t/c ratio were analyzed in both Young and Aged beer samples. Total polyphenol content generally increased in both young and aged treatments compared to controls. Analysis of the t/c ratio suggests that both Young and Aged beers were chemically preserved to some degree after approximately 12 h at the given dry hopping rate regardless of age. Within the Aged beer trials, 96 h of dry hop contact yielded a significant increase in t/c ratio compared to all other Aged trials. This suggests that a 4-day dry hop regime may yield additional oxidative protection of iso-α-acids in beers stored unrefrigerated for 30 days. Descriptive analysis was also performed with an 8-person, trained panel; however, beers were sensorially distinguished by their aging time as opposed to their dry hopping time.

Linoleic acid-derived 13-hydroxyoctadecadienoic acid is absorbed and incorporated into rat tissues.

Linoleic acid (LNA)-derived 13-hydroxyoctadecadienoic acid (13-HODE) is a bioactive lipid mediator that regulates multiple signaling processes in vivo. 13-HODE is also produced when LNA is oxidized during food processing. However, the absorption and incorporation kinetics of dietary 13-HODE into tissues is not known. The present study measured unesterified d4-13-HODE plasma bioavailability and incorporation into rat liver, adipose, heart and brain following gavage or intravenous (IV) injection (n = 3 per group). Mass spectrometry analysis revealed that d4-13-HODE was absorbed within 20 min of gavage, and continued to incorporate into plasma esterified lipid fractions throughout the 90 min monitoring period (incorporation half-life of 71 min). Following IV injection, unesterified d4-13-HODE was rapidly eliminated from plasma with a half-life of 1 min. Analysis of tracer incorporation kinetics into rat tissues following IV injection or gavage revealed that the esterified tracer preferentially incorporated into liver, adipose and heart compared to unesterified d4-13-HODE. No tracer was detected in the brain. This study demonstrates that dietary 13-HODE is absorbed, and incorporated into peripheral tissues from esterified plasma lipid pools. Understanding the chronic effects of dietary 13-HODE exposure on peripheral tissue physiology and metabolism merits future investigation.

Influence of post-harvest moisture on roasted almond shelf life and consumer acceptance.

BACKGROUND: The harvest weights of sweet almonds (Prunus dulcis) have significantly increased to meet consumer demand and now exceed processing facility capabilities. Crops are stockpiled for longer periods, increasing the probability of moisture exposure. Wet almonds can be mechanically dried prior to processing; however, it is unclear how this practice influences lipid oxidation, shelf-life, and consumer acceptance. To address this, almonds were exposed to 8% moisture and dried with low heat (ME). Almonds were roasted and stored under accelerated conditions for 12 months and markers of lipid oxidation, headspace volatiles, sensory attributes, and consumer liking were evaluated. RESULTS: At 7 months of storage, light roast ME almonds had higher levels of volatiles related to lipid oxidation than non-moisture exposed almonds (NME) and were significantly higher in oxidized, cardboard and painty / solvent flavors. Although untrained consumers did not show significant preferences between the light roast ME and NME almonds, there were quality losses related to lipid oxidation that trained panelists could detect. Dark roast ME almonds demonstrated significant lipid oxidation by 5 months of storage, indicating they will have a compromised shelf life. Findings also indicate that octanal, nonanal, 2-octenal, and hexanoic acid are good indicators of consumer acceptability. CONCLUSION: The results of this research illustrate that post-harvest moisture exposure with mechanical drying has a significant effect on the storage quality of roasted almonds and is most pronounced in dark roast products. © 2020 Society of Chemical Industry.

Evaluation of the Intrinsic and Perceived Quality of Sangiovese Wines from California and Italy.

Sangiovese is the most cultivated red grape variety in Italy where it is certified for the production of several Protected Designation of Origin (PDO) wines, and it is one of the most cultivated Italian red grape varieties in California. Despite the global distribution of this variety, there is a lack of international studies on Sangiovese grapes and wines. For this reason, the present study aimed to compare 20 commercial Sangiovese wines from 2017 harvest, 9 produced in Italy (Tuscany) and 11 in California, in order to evaluate the intrinsic and perceived quality. The eligibility, identity, and style properties (the intrinsic quality) of the wines were evaluated. A group of 11 Italian experts evaluated the perceived quality by rating the typicality of the wines. The experimental data showed that the intrinsic quality of Sangiovese wine samples was affected by the growing area; in particular, the wine resulted very different for the color indices and polyphenol composition. The above differences in intrinsic quality levels did not lead to a different evaluation of the perceived quality (typicality) by the wine experts. The results evidenced that Sangiovese variety is recognizable also if grown outside its original terroir, and fresh and fruity wines were considered more typical. This study expands our current knowledge of Sangiovese wines and the contribution of regional characteristics to the composition of wine.

Impact of Grapevine Red Blotch Disease on Cabernet Sauvignon and Merlot Wine Composition and Sensory Attributes.

Grapevine red blotch disease (GRBD) is a recently identified viral disease that affects grapevines. GRBD has been shown to impact grapevine physiology and grape composition by altering specific ripening events. However, no studies have been reported on the impact of GRBD on wine composition and its sensory attributes. This study evaluated the impact of GRBD on wine primary and secondary metabolites, in addition to its sensory properties, when making wines from Cabernet Sauvignon and Merlot grapes during two seasons. Wines made with GRBD-impacted fruit were lower in ethanol content when compared to wines made with grapes from healthy grapevines. This was attributed to the lower total soluble sugar (TSS) levels of diseased grapes due to delayed ripening at harvest. GRBD impacted wine phenolic composition by decreasing anthocyanin concentrations and increasing flavonol concentrations in some instances. Additionally, proanthocyanidin concentrations were also consistently higher in GRBD wines compared to wines made from healthy fruit. Descriptive analysis demonstrated that GRBD can impact wine style by altering aroma, flavor, and mouthfeel attributes. However, the extent of GRBD impact on wine composition and sensory properties were site and season dependent.

Direct Analysis of Glycosidic Aroma Precursors Containing Multiple Aglycone Classes in Vitis vinifera Berries.

Ultra-high-performance liquid chromatography (UHPLC) accurate mass tandem mass spectrometry is a powerful tool for identifying and profiling plant metabolites. Here, we describe an approach to characterize glycosidically bound precursors of monoterpenoids, norisoprenoids, volatile phenols, aliphatic alcohols, and sesquiterpenoids in grapes. Chromatographic separation of glycosylated compounds was evaluated using phenyl-hexyl (reverse phase), glycan/hydrophilic interaction, and porous graphitic carbon (PGC) stationary phases. PGC provided the best UHPLC separation for 102 tentatively identified aroma precursors in Vitis vinifera L. cv. Riesling and Muscat of Alexandria berries. Monoterpene-triol, monoterpene-tetraol, and sesquiterpenol glycosides were tentatively identified for the first time in grapes, and a C6-alcohol trisaccharide was tentatively identified for the first time in any plant. Comparison of glycosylated aroma molecules in Riesling and Muscat of Alexandria grapes showed that the two varieties were distinguishable based on relative abundances of shared glycosides and the presence of glycosides unique to a single variety.

Impact of Grapevine Red Blotch Disease on Grape Composition of Vitis vinifera Cabernet Sauvignon, Merlot, and Chardonnay.

Grapevine red blotch disease (GRBD) is a recently recognized viral disease that affects grapevines ( Vitis vinifera L.). Currently little is known about its impact on grape composition. This study focused on the impact of GRBD on grape primary and secondary metabolites (mainly phenolic compounds) of three Vitis vinifera L. cultivars during two seasons. Grapes from symptomatic red blotch diseased vines (RB (+)) mostly had lower concentration of total soluble solids (TSS) and higher titratable acidity (TA) levels when compared to grapes from healthy vines (RB (-)) at harvest. GRBD impacted grape phenolic composition by mostly decreasing anthocyanin and increasing flavonol and proanthocyanidin (PA) contents in berry skins. No major impacts were observed on seed phenolics. RB (+) grapes contained more amino and carboxylic acids, while RB (-) grapes contained more oligosaccharides, polyols, and some specific monosaccharides at harvest. The impact of GRBD on grape composition was variable and dependent on the cultivar, site, and season.

Chemical Characteristics of Sangiovese Wines from California and Italy of 2016 Vintage.

Sangiovese is the most widespread Italian red cultivar and constitutes the basis of internationally known wines such as Chianti and Brunello di Montalcino. Outside of Europe, Argentina is the largest producer, followed by the United States. This study sought to define and compare 2016 vintage Sangiovese wine composition from various production regions in California and Italy. Forty-six commercial Sangiovese wines from California and Italy were analyzed for volatile profile, color, phenolic, and elemental content. This study demonstrates that it is possible to determine regional differences among wines based on these chemical profiles. However, some Californian and Italian wine had similar chemical compositions. In order to compare Californian and Italian wines, Californian wine reference models were developed using the chemical parameters from Sangiovese wines, performing a Soft Independent Modeling of Class Analogy (SIMCA). To our knowledge, this is the first time that an extensive regionality study has been attempted for Sangiovese wines.

Impact of cold soak duration on Cabernet Sauvignon fermentation and phenolic composition.

BACKGROUND: Cold soak is a prefermentative maceration technique believed to enhance grape skin extraction. Studies show variable results depending on cold soak and winemaking conditions. To investigate the effect of cold soak more fully, systematic and highly reproducible Cabernet Sauvignon fermentations with increasing cold-soak durations were performed. RESULTS: Phenolic extraction during cold soak and fermentation showed significant differences among all treatments for monitored phenolics at the end of the cold soak. At the end of alcoholic fermentation only gallic acid, (-)-epicatechin, and the flavonols were significant, and only (-)-epicatechin was significant after bottle ageing. Descriptive analysis of the bottled wines showed that the 4- and 7-day treatments were significantly higher in caramelized/vanilla/browned flavor compared to the 1-day treatment and lower levels of bitterness were observed up to 2 days of cold soak. While oligosaccharide content increased with increasing cold-soak duration, differences were not large enough to result in sensory differences. CONCLUSION: While increased cold soak duration led to differences in phenolic extraction during early fermentation, these differences did not last through to the end product. Thus, under the conditions of this study, cold-soak duration had little overall impact on Cabernet Sauvignon wine composition and style. © 2018 Society of Chemical Industry.

Characterization of Red Wine Proanthocyanidins Using a Putative Proanthocyanidin Database, Amide Hydrophilic Interaction Liquid Chromatography (HILIC), and Time-of-Flight Mass Spectrometry.

Proanthocyanidins are complex polymers of flavan-3-ol monomers and play a key sensory and health role in foods and beverages. We describe here a novel method for characterizing wine proanthocyanidins using a theoretical database comprised of the chemical formula and exact mass of 996 compounds. The database was constructed using the four primary grape and wine proanthocyanidin monomers: (epi)catechin, (epi)catechin-3-O-gallate, (epi)gallocatechin, and (epi)gallocatechin-3-O-gallate, each combined in all possible combinations up to a polymerization of 10. The database was queried against spectra collected using ultrahigh performance liquid chromatography (UHLPC) with a hydrophilic interaction liquid chromatography (HILIC) column and coupled to a high-resolution accurate mass quadrupole time-of-flight mass spectrometer (Q-TOF MS). Two wine samples produced with different post fermentation maceration were analyzed using the presented method to demonstrate application for analysis of diverse proanthocyanidins. The first sample was pressed immediately at the end of fermentation when all sugar had been utilized and the second received eight weeks of post fermentation maceration. The HILIC column combined with high resolution tandem mass spectrometry and database matching provided tentative identification of 89 compounds with excellent resolution and without the need for two-dimensional separations. The identified compounds were visualized with Kendrick mass analysis, a simple technique allowing for rapid visualization of which compounds are present in a given sample.

Investigating the Effect of Cold Soak Duration on Phenolic Extraction during Cabernet Sauvignon Fermentation.

The impact of increasing cold soak (CS) duration (0, 1, 4, 7, and 10 days at 10 °C) on the extraction of phenolic compounds during the CS period and primary fermentation as well as the final composition of Cabernet Sauvignon wine was investigated. The results showed that CS duration had no effect on hydroxycinnamate and flavonol extractions. Greater amounts of gallic acid, (+)-catechin, (-)-epicatechin, and total tannins were extracted with increasing CS duration, with differences maintained during bottle aging. Anthocyanin extraction and color density increased with longer periods of CS; however, by the end of primary fermentation, as well as three months' bottle aging, there were no significant differences due to CS duration. The wines made with seven and 10 days of CS had higher seed tannin contributions and total tannin compared to the non-CS wine, which could potentially result in increased astringency.

Fermentation temperature modulates phosphatidylethanolamine and phosphatidylinositol levels in the cell membrane of Saccharomyces cerevisiae.

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at "normal" temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner.

Multiple precursor ion scanning of gangliosides and sulfatides with a reversed-phase microfluidic chip and quadrupole time-of-flight mass spectrometry.

Precise profiling of polar lipids including gangliosides and sulfatides is a necessary step in understanding the diverse physiological role of these lipids. We have established an efficient method for the profiling of polar lipids using reversed-phase nano high-performance liquid chromatography microfluidic chip quadrupole time-of-flight mass spectrometry (nano-HPLC-chip Q-TOF/MS). A microfluidic chip design provides improved chromatographic performance, efficient separation, and stable nanospray while the advanced high-resolution mass spectrometer allowed for the identification of complex isobaric polar lipids such as NeuAc- and NeuGc-containing gangliosides. Lipid classes were identified based on the characteristic fragmentation product ions generated during data-dependent tandem mass spectrometry (MS/MS) experiments. Each class was monitored by a postprocessing precursor ion scan. Relatively simple quantitation and identification of intact ions was possible due to the reproducible retention times provided by the nano-HPLC chip. The method described in this paper was used to profile polar lipids from mouse brain, which was found to contain 17 gangliosides and 13 sulfatides. Types and linkages of the monosaccharides and their acetyl modifications were identified by low-energy collision-induced dissociation (CID) (40 V), and the type of sphingosine base was identified by higher energy CID (80 V). Accurate mass measurements and chromatography unveiled the degree of unsaturation and hydroxylation in the ceramide lipid tails.

Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry.

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.

Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk gangliosides was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.

Nano-LC-MS/MS of glycopeptides produced by nonspecific proteolysis enables rapid and extensive site-specific glycosylation determination.

Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopeptides and the ability to determine glycan composition and site-specific glycosylation. However, because of the size of glycopeptides, they are not often amenable to tandem MS. In this work, proteins are digested with multiple proteases to produce glycopeptides that are of suitable size for tandem MS analysis. The conditions for collision-induced dissociation are optimized to obtain diagnostic ions that maximize glycan and peptide information. The method is applied to glycoproteins with contrasting glycans and multiple sites of glycosylation and identifies multiple glycan compositions at each individual glycosylation site. This method provides an important improvement in the routine determination of glycan microheterogeneity by mass spectrometry.