Bondoc, K. G., et al. (2013). “Chemical fingerprinting and phylogenetic mapping of saponin congeners from three tropical holothurian sea cucumbers.” Comp Biochem Physiol B Biochem Mol Biol 166(3-4): 182-193.
Holothurians are sedentary marine organisms known to produce saponins (triterpene glycosides), secondary metabolites exhibiting a wide range of biological activities. In this paper, we investigated the saponin contents of semi-purified and membranolytic HPLC fractionated extracts from the body wall of three species of Holothuriidae as an attempt to examine its chemical diversity in relation to phylogenetic data. MALDI-FTICR MS and nano-HPLC-chip Q-TOF MS were used for mass profiling and isomer separation, respectively giving a unique chemical saponin fingerprint. Moreover, the methods used yield the highest number of congeners. However, saponin concentration, bioactivity and chemical diversity had no apparent relationship. MS fingerprint showed the presence of holothurinosides, which was observed for the first time in other Holothuria genera besides the basally positioned Holothuria forskali. This congener is proposed to be a primitive character that could be used for taxonomic purposes. The phylogenetic mapping also showed that the glycone part of the compound evolved from non-sulfated hexaosides to sulfated tetraosides, which have higher membranolytic activity and hydrophilicity, the two factors affecting the total ecological activity (i.e. chemical defense) of these compounds. This might be an adaptation to increase the fitness of the organism.

Guerrero, A. and C. B. Lebrilla (2013). “New strategies for resolving oligosaccharide isomers by exploiting mechanistic and thermochemical aspects of fragment ion formation.” Int J Mass Spectrom 354-355.
Three complementary experimental approaches for elucidating human milk oligosaccharide (HMOs) isomers by Fourier Transform Ion Cyclotron Resonance mass spectrometry (FT-ICR) are described: tandem-MS disruption by double resonance to distinguish different fragmentation pathways, examination of fragment intensity ratios arising from differential alkali metal ion affinities and monitoring competitive fragmentation rates. The interpretation of the fragmentation pattern from a mechanistic and thermochemical point of view permits the assignment of not only pure isomers but, in some cases, mixtures of them. Methodologically the procedures are simple, reliable and rapid making unnecessary both the use of previous separation techniques and tedious chemical modifications of the HMOs. In principle, the rationale can be expanded to resolve other isomeric mixtures of biological nature.

Hong, Q., et al. (2013). “Absolute quantitation of immunoglobulin G and its glycoforms using multiple reaction monitoring.” Anal Chem 85(18): 8585-8593.
Studies aimed toward glycan biomarker discovery have focused on glycan characterization by the global profiling of released glycans. Site-specific glycosylation analysis is less developed but may provide new types of biomarkers with higher sensitivity and specificity. Quantitation of peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with specific proteins at distinct sites. We have developed a method using MRM to monitor protein glycosylation normalized to absolute protein concentrations to examine quantitative changes in glycosylation at a site-specific level. This new approach provides information regarding both the absolute amount of protein and the site-specific glycosylation profile and will thus be useful to determine if altered glycosylation profiles in serum/plasma are due to a change in protein glycosylation or a change in protein concentration. The remarkable sensitivity and selectivity of MRM enable the detection of low abundance IgG glycopeptides, even when IgG was digested directly in serum with no cleanup prior to the liquid chromatography. Our results show a low limit of detection of 60 amol and a wide dynamic range of 3 orders magnitude for IgG protein quantitation. The results show that IgG glycopeptides can be analyzed directly from serum (without enrichment) and yield more accurate abundances when normalized to the protein content. This report represents the most comprehensive study so far of the use of multiple reaction monitoring for the quantitation of glycoproteins and their glycosylation patterns in biofluids.

Hua, S., et al. (2013). “Glyco-analytical multispecific proteolysis (Glyco-AMP): a simple method for detailed and quantitative Glycoproteomic characterization.” J Proteome Res 12(10): 4414-4423.
Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.

Hua, S., et al. (2013). “Differentiation of Cancer Cell Origin and Molecular Subtype by Plasma Membrane N-Glycan Profiling.” J Proteome Res.
In clinical settings, biopsies are routinely used to determine cancer type and grade based on tumor cell morphology, as determined via histochemical or immunohistochemical staining. Unfortunately, in a significant number of cases, traditional biopsy results are either inconclusive or do not provide full subtype differentiation, possibly leading to inefficient or ineffective treatment. Glycomic profiling of the cell membrane offers an alternate route towards cancer diagnosis. In this study, isomer-sensitive nano-LC/MS was used to directly obtain detailed profiles of the different N-glycan structures present on cancer cell membranes. Membrane N-glycans were extracted from cells representing various subtypes of breast, lung, cervical, ovarian, and lymphatic cancer. Chip-based porous graphitized carbon nano-LC/MS was used to separate, identify, and quantify the native N-glycans. Structure-sensitive N-glycan profiling identified hundreds of glycan peaks per cell line, including multiple isomers for most compositions. Hierarchical clusterings based on Pearson correlation coefficients were used to quickly compare and separate each cell line according to originating organ and disease subtype. Based simply on the relative abundances of broad glycan classes (e.g. high mannose, complex/hybrid fucosylated, complex/hybrid sialylated, etc.) most cell lines were readily differentiated. More closely-related cell lines were differentiated based on several-fold differences in the abundances of individual glycans. Based on characteristic N-glycan profiles, primary cancer origins and molecular subtypes could be distinguished. These results demonstrate that stark differences in cancer cell membrane glycosylation can be exploited to create an MS-based biopsy, with potential applications towards cancer diagnosis and direction of treatment.

Kailemia, M. K., et al. (2013). “Oligosaccharide Analysis By Mass Spectrometry: A Review Of Recent Developments.” Anal Chem.
This review covers developments in the application of mass spectrometry to the analysis of carbohydrates, focusing on work published between January 2011 through October 2013. These developments include approaches for improved ionization, new and improved methods of ion activation, advances in chromatographic separations of carbohydrates, the hybridization of ion mobility and mass spectrometry, and better software for data collection and interpretation. The impact of the developments are considered in terms of their ability to solve new problems or provide new capabilities for carbohydrate analysis, particularly for structure characterization by mass spectrometry and tandem mass spectrometry.

Lee, H., et al. (2013). “Quantitative analysis of gangliosides in bovine milk and colostrum-based dairy products by ultrahigh performance liquid chromatography-tandem mass spectrometry.” J Agric Food Chem 61(40): 9689-9696.
Milk gangliosides have gained considerable attention because they participate in diverse biological processes, including neural development, pathogen binding, and activation of the immune system. Herein, we present a quantitative measurement of the gangliosides present in bovine milk and other dairy products and byproducts. Ultrahigh performance liquid chromatography separation was used for high-throughput analysis and achieved a short running time without sacrificing chromatographic resolution. Dynamic multiple reaction monitoring was conducted for 12 transitions for GM3 and 12 transitions for GD3. Transitions to sialic acid fragments (m/z 290.1) were chosen for the quantitation. There was a considerable amount of gangliosides in day 2 milk (GM3, 0.98 mg/L; GD3, 15.2 mg/L) which dramatically decreased at day 15 and day 90. GM3 and GD3 were also analyzed in pooled colostrum, colostrum cream, colostrum butter, and colostrum buttermilk. The separation and analytical approaches here proposed could be integrated into the dairy industry processing adding value to side-streams.

Ozcan, S., et al. (2013). “Serum glycan signatures of gastric cancer.” Cancer Prev Res (Phila).
Glycomics, a comprehensive study of glycans expressed in biological systems, is emerging as a simple yet highly sensitive diagnostic tool for disease onset and progression. This study aimed to use glycomics to investigate glycan markers that would differentiate patients with gastric cancer (GC) from those with non-atrophic gastritis (NAG). Patients with duodenal ulcer (DU) were also included because they are thought to represent a biologically different response to infection with Helicobacter pylori, a bacterial infection that can cause either GC or DU. We collected 72 serum samples from patients in Mexico City that presented with NAG, DU, or GC. N-glycans were released from serum samples using the generic method with PNGase F and were analyzed by MALDI FT-ICR MS. The corresponding glycan compositions were calculated based on accurate mass. ANOVA based statistical analysis was performed to identify potential markers for each sub-group. Nineteen glycans were significantly different among the diagnostic groups. Generally, decreased levels of high-mannose type glycans, glycans with one complex type antenna, bigalactosylated biantennary glycans, and increased levels of non-galactosylated biantennary glycans were observed in gastric cancer cases. Altered levels of serum glycans were also observed in DU, but differences were generally in the same direction as GC. Serum glycan profiles may provide biomarkers to differentiate GC cases from controls with NAG. Further studies will be needed to validate these findings as biomarkers and identify the role of protein glycosylation in GC pathology.

Peacock, K. S., et al. (2013). “Isomer-Specific Consumption of Galactooligosaccharides by Bifidobacterial Species.” J Agric Food Chem.
Prebiotics are nondigestible substrates that stimulate the growth of beneficial microbes in the human intestine. Galactooligosaccharides (GOS) are food ingredients that possess prebiotic properties, in particular, promoting the growth of bifidobacteria in situ. However, precise mechanistic details of GOS consumption by bifidobacteria remain poorly understood. Because GOS are mixtures of polymers of different lengths and linkages, there is interest in determining which specific structures provide prebiotic effects to potentially create better supplements. This paper presents a method comprising porous graphitic carbon separation, isotopic labeling, and mass spectrometry analysis for the structure-specific analysis of GOS isomers and their bacterial consumption rate. Using this strategy, the differential bacterial consumption of GOS by the bifidobacteria species Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, and Bifidobacterium adolescentis was determined, indicating that the use of specific GOS isomers in infant formula may provide enrichment of distinct species.

Smilowitz, J. T., et al. (2013). “Human milk secretory immunoglobulin a and lactoferrin N-glycans are altered in women with gestational diabetes mellitus.” J Nutr 143(12): 1906-1912.
Very little is known about the effects of gestational diabetes mellitus (GDM) on lactation and milk components. Recent reports suggested that hyperglycemia during pregnancy was associated with altered breast milk immune factors. Human milk oligosaccharides (HMOs) and N-glycans of milk immune-modulatory proteins are implicated in modulation of infant immunity. The objective of the current study was to evaluate the effect of GDM on HMO and protein-conjugated glycan profiles in breast milk. Milk was collected at 2 wk postpartum from women diagnosed with (n = 8) or without (n = 16) GDM at week 24-28 in pregnancy. Milk was analyzed for HMO abundances, protein concentrations, and N-glycan abundances of lactoferrin and secretory immunoglobulin A (sIgA). HMOs and N-glycans were analyzed by mass spectrometry and milk lactoferrin and sIgA concentrations were analyzed by the Bradford assay. The data were analyzed using multivariate modeling confirmed with univariate statistics to determine differences between milk of women with compared with women without GDM. There were no differences in HMOs between milk from women with vs. without GDM. Milk from women with GDM compared with those without GDM was 63.6% lower in sIgA protein (P < 0.05), 45% higher in lactoferrin total N-glycans (P < 0.0001), 36-72% higher in lactoferrin fucose and sialic acid N-glycans (P < 0.01), and 32-43% lower in sIgA total, mannose, fucose, and sialic acid N-glycans (P < 0.05). GDM did not alter breast milk free oligosaccharide abundances but decreased total protein and glycosylation of sIgA and increased glycosylation of lactoferrin in transitional milk. The results suggest that maternal glucose dysregulation during pregnancy has lasting consequences that may influence the innate immune protective functions of breast milk.

Underwood, M. A., et al. (2013). “Prebiotic Oligosaccharides In Premature Infants.” J Pediatr Gastroenterol Nutr.
OBJECTIVE:: To determine the impact of increasing doses of two prebiotic oligosaccharides and the impact of an “all-human diet” on the intestinal microbiota of premature infants. METHODS:: Twelve premature infants receiving formula feedings were randomly assigned to receive either galacto-oligosaccharide (F+GOS) or a pooled concentrated donor human milk product containing human milk oligosaccharides (F+HMO) in increasing doses over a five week period. A second group of fifteen premature infants received their mother’s own milk fortified with either a concentrated donor human milk product (H+H) or a bovine powdered fortifier (H+B). Serial stool specimens from each infant were analyzed by terminal restriction fragment length polymorphism and quantitative polymerase chain reaction for bacterial composition. RESULTS:: All infants studied had relatively low levels of bifidobacteria and no measurable Lactobacilli. Infants in the F+GOS and F+HMO groups demonstrated an increase in relative numbers of Clostridia with increasing doses. Compared to the H+B group, the infants in the F+HMO and the H+H groups showed an unexpected trend towards an increase in gamma-Proteobacteria over time/dose. Principal Coordinate Analyses and Shannon diversity scores were not significantly different among the four groups. Infants in the H+H group received more antibiotics during the study period than those in the other groups. Two of the infants receiving GOS developed feeding intolerance. CONCLUSIONS:: None of the prebiotic interventions resulted in significant increases in bifidobacteria compared to baseline specimens or to the H+B group, however many of the infants did not receive the highest doses of GOS and HMO and antibiotic use in the H+H group was high.