Larry A. Lerno

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.

Bifidobacterium longum subsp. infantis ATCC 15697 α-Fucosidases Are Active on Fucosylated Human Milk Oligosaccharides

ABSTRACT 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.

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.

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.

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.

Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae

ABSTRACT 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.

Barrel maturation, oak alternatives and micro-oxygenation: Influence on red wine aging and quality

The impact of micro-oxygenation (MOX) in conjunction with a variety of oak alternatives on phenolic composition and red wine aging was investigated and compared with traditional barrel aging. Although several studies concluded that MOX give similar results to barrel aging, few have compared them directly and none directly compared MOX with and without wood alternatives and barrel aging. Results confirmed that MOX had a positive effect on colour density, even after 5 months of bottle aging. This is supported by an increase in polymeric phenol and pigment content not only with aging but in the MOX compared to barrel matured wine treatments. Descriptive analysis showed that MOX in combination with wood alternatives such as oak chips and staves could mimic short term (six months) barrel aging in new American and French oak barrels in regards to sensory characteristics.

Method for the Identification of Lipid Classes Based on Referenced Kendrick Mass Analysis

A rapid method for the determination of lipid classes with high sensitivity is described. The referenced Kendrick mass defect (RKMD) and RKMD plots are novel adaptations of the Kendrick mass defect analysis that allows for the rapid identification of members of a homologous series in addition to identifying the lipid class. Assignment of lipid classes by the RKMD method is accomplished by conversion of the lipid masses to the Kendrick mass scale and then referencing the converted masses to each lipid class. Referencing of the masses to a given lipid class is achieved by first subtracting the heteroatom and lipid backbone contributions to the mass defect, leaving behind the contribution to the mass by the fatty acid constituents. The final step in the referencing makes use of spacing differences in mass defects between members of the same Kendrick class to identify members of the lipid class being referenced. The end result of this is that a lipid belonging to the class being referenced will have an integer RKMD with the value of the integer being the degrees of unsaturation in the lipid. The RKMD method was able to successfully identify the lipids in an idealized data set consisting of 160 lipids drawn from the glyceride and phosphoglyceride classes. As a real world example the lipid extract from bovine milk was analyzed using both accurate mass measurements and the RKMD method.

Effects of Cap and Overall Fermentation Temperature on Phenolic Extraction in Cabernet Sauvignon Fermentations

The phenolic content of red wine is responsible for the color, mouthfeel, and aging potential of the wine. Although many fermentation parameters and winemaking techniques affect phenolic extraction, it is generally agreed that one of the prime factors is fermentation temperature; however, temperature is not uniform during fermentation and thermal gradients form between the cap and the liquid. To determine the effects of temperature on phenolic extraction, research scale (120 L) Cabernet Sauvignon fermentations were performed in which the cap and must were either maintained at the same temperature or a constant thermal gradient was maintained between the two during the period of active fermentation. All fermentations were sampled twice daily and phenolic content was determined by reverse-phase high-performance liquid chromatography for the monomeric phenolics and UV-vis spectroscopy for the total anthocyanins and condensed tannins. These experiments showed that cap and must temperature had noticeable effects on phenolic extraction based on where the phenolics originated. For skin phenolics, temperature affected the rate of extraction but not the final concentration, and increasing temperatures favored faster extraction. For seed phenolics, increases in fermentation temperature increased both the rate of extraction and the final concentration. Results showed that must temperature was more important than cap temperature in driving extraction of phenolics.

Top-Down Analysis of Highly Post-Translationally Modified Peptides by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

AbstractBovine κ-caseinoglycomacropeptide (GMP) is a highly modified peptide from κ-casein produced during the cheese making process. The chemical nature of GMP makes analysis by traditional proteomic approaches difficult, as the peptide bears a strong net negative charge and a variety of post-translational modifications. In this work, we describe the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) for the top-down analysis of GMP. The method allows the simultaneous detection of different GMP forms that result from the combination of amino acid genetic variations and post-translational modifications, specifically phosphorylation and O-glycosylation. The different GMP forms were identified by high resolution mass spectrometry in both negative and positive mode and confirmation was achieved by tandem MS. The results showed the predominance of two genetic variants of GMP that occur as either mono- or bi-phosphorylated species. Additionally, these four forms can be modified with up to two O-glycans generally sialylated. The results demonstrate the presence of glycosylated, bi-phosphorylated forms of GMP never described before. Graphical Abstractᅟ