Investigating Grapevine Red Blotch Virus Infection in Vitis vinifera L. cv. Cabernet Sauvignon Grapes: A Multi-Omics Approach.

Grapevine red blotch virus (GRBV) is a recently identified virus. Previous research indicates primarily a substantial impact on berry ripening in all varieties studied. The current study analyzed grapes' primary and secondary metabolism across grapevine genotypes and seasons to reveal both conserved and variable impacts to GRBV infection. Vitis vinifera cv. Cabernet Sauvignon (CS) grapevines grafted on two different rootstocks (110R and 420A) were analyzed in 2016 and 2017. Metabolite profiling revealed a considerable impact on amino acid and malate acid levels, volatile aroma compounds derived from the lipoxygenase pathway, and anthocyanins synthesized in the phenylpropanoid pathway. Conserved transcriptional responses to GRBV showed induction of auxin-mediated pathways and photosynthesis with inhibition of transcription and translation processes mainly at harvest. There was an induction of plant-pathogen interactions at pre-veraison, for all genotypes and seasons, except for CS 110R in 2017. Lastly, differential co-expression analysis revealed a transcriptional shift from metabolic synthesis and energy metabolism to transcription and translation processes associated with a virus-induced gene silencing transcript. This plant-derived defense response transcript was only significantly upregulated at veraison for all genotypes and seasons, suggesting a phenological association with disease expression and plant immune responses.

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.

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.

Impact of Temperature, Ethanol and Cell Wall Material Composition on Cell Wall-Anthocyanin Interactions.

The effects of temperature and ethanol concentration on the kinetics of anthocyanin adsorption and desorption interactions with five cell wall materials (CWM) of different composition were investigated. Using temperatures of 15 °C and 30 °C and model wine with ethanol concentrations of 0% and 15% (v/v) over 120 min, the adsorption and desorption rates of five anthocyanin-glucosides were recorded in triplicate. Small-scale experiments were conducted using a benchtop incubator to mimic a single berry fermentation. Results indicate that more than 90% of the adsorption occurs within the first 60 min of the addition of anthocyanins to CWM. However, desorption appears to occur much faster, with maximum desorption being reached after 30 min. The extent of both adsorption and desorption was clearly dependent not only on temperature and ethanol concentration but also on the CWM composition.

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.

Discovery of Potent Glycosidases Enables Quantification of Smoke-Derived Phenolic Glycosides through Enzymatic Hydrolysis.

When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

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.

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.

An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides.

Lactating mothers secrete milk sialyloligosaccharides (MSOs) that function as anti-adhesives once provided to the neonate. Particular infant-associated commensals, such as Bifidobacterium longum subsp. infantis, consume neutral milk oligosaccharides, although their ability to utilize acidic oligosaccharides has not been assessed. Temporal glycoprofiling of acidic HMO consumed during fermentation demonstrated a single composition, with several isomers, corresponding to sialylated lacto-N-tetraose. To utilize MSO, B. longum subsp. infantis deploys a sialidase that cleaves α2-6 and α2-3 linkages. NanH2, encoded within the HMO catabolic cluster is up-regulated during HMO fermentation and is active on sialylated lacto-N-tetraose. These results demonstrate that commensal microorganisms do utilize MSO, a substrate that may be enriched in the distal gastrointestinal tract.

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.

Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides.

Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 α-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 α-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.

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.

Extraction and Analysis of Phenolic Compounds from Grape Berries.

Phenolics are ubiquitous compounds that represent the most abundant and diverse class of plant metabolites. From an analytical point of view, phenolics can be divided into soluble phenolics such as phenolic acids, phenylpropanoids, flavonoids and quinones, and nonsoluble compounds such as proanthocyanidins, lignins, and cell wall-bound hydroxycinnamic acids. Extraction of phenolics from the sample material is the first step toward their analysis. Biochemical methods for determination of total phenolics content were widely used in the past but modern chromatographic and mass spectrometric methods for identification and quantification of individual compounds are available in recent years. In this chapter, we describe methods for phenolic compounds extraction used in our laboratories from berries of Vitis vinifera and analytical methods including HPLC coupled to DAD detector and Q-TOF LC/MS for their analysis.

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.

A Field Collection of Indigenous Grapevines as a Valuable Repository for Applied Research.

The grapevine is an economically important plant, with a historical connection to the development of human culture. Currently, over 6000 accessions are known as individual grapevine varieties, some of which are important to national heritage, valuable for current viticultural practices, and as genetic resources to maintain plasticity under changing climatic conditions, environmental sustainability, and market demands. Recently, the diversity of cultivated grapevines has declined significantly, due to the increased focus of global wine industries on a few major cultivars. Moreover, due to biotic and abiotic stresses, the wild V. vinifera germplasm's genetic diversity has declined, with some varieties on the verge of extinction. Vitis germplasm conservation can be achieved via either in situ (e.g., protected areas) or Ex situ (e.g., field collections, seed banks, and tissue culture collections) methods. This study aims to highlight the importance of Vitis field bank collections. We demonstrate the research done in the Israeli indigenous Vitis vinifera collection. The multi-layer analysis of the varieties enabled the identification of drought stress-resistant varieties, and suggested a mechanism for this resistance through noting the dramatic phenological differences in foliage development between resistant and sensitive varieties. In addition, we show a general characterization of the varieties via major grape characteristics, including bunch and berry shape, as well as their possible utilization based on their aromatic and phenolic profiles.

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.

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.