Profiling Intact Glycosphingolipids with Automated Structural Annotation and Quantitation from Human Samples with Nanoflow Liquid Chromatography Mass Spectrometry.

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome. The high sensitivity obtained using nanorange flow rates with robust chromatographic reproducibility over a wide range of concentrations and injection volumes results in confident identifications for profiling these low-abundant biomolecules.

Evaluating the use of locked nucleic acid capture probes in hybrid LC-MS/MS analysis of siRNA analytes.

Background: Hybrid LC-MS assays for oligonucleotides rely on capture probes to develop assays with high sensitivity and specificity. Locked nucleic acid (LNA) probes are thermodynamically superior to existing capture probes, but are not currently used for hybrid LC-MS assays. Materials & methods: Using two lipid-conjugated double-stranded siRNA compounds as model analytes, hybrid LC-MS/MS assays using LNA probes were developed. Results: The workflows demonstrated the superiority of the LNA probes, optimized sample preparation conditions to maximize analyte recovery, evaluated the need for analyte-specific internal standards, and demonstrated that advanced mass spectrometric technology can increase assay sensitivity by up to 20-fold. Conclusion: The workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for siRNA analytes.

Transcriptomic and glycomic analyses highlight pathway-specific glycosylation alterations unique to Alzheimer's disease.

Glycosylation has been found to be altered in the brains of individuals with Alzheimer's disease (AD). However, it is unknown which specific glycosylation-related pathways are altered in AD dementia. Using publicly available RNA-seq datasets covering seven brain regions and including 1724 samples, we identified glycosylation-related genes ubiquitously changed in individuals with AD. Several differentially expressed glycosyltransferases found by RNA-seq were confirmed by qPCR in a different set of human medial temporal cortex (MTC) samples (n = 20 AD vs. 20 controls). N-glycan-related changes predicted by expression changes in these glycosyltransferases were confirmed by mass spectrometry (MS)-based N-glycan analysis in the MTC (n = 9 AD vs. 6 controls). About 80% of glycosylation-related genes were differentially expressed in at least one brain region of AD participants (adjusted p-values < 0.05). Upregulation of MGAT1 and B4GALT1 involved in complex N-linked glycan formation and galactosylation, respectively, were reflected by increased concentrations of corresponding N-glycans. Isozyme-specific changes were observed in expression of the polypeptide N-acetylgalactosaminyltransferase (GALNT) family and the alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase (ST6GALNAC) family of enzymes. Several glycolipid-specific genes (UGT8, PIGM) were upregulated. The critical transcription factors regulating the expression of N-glycosylation and elongation genes were predicted and found to include STAT1 and HSF5. The miRNA predicted to be involved in regulating N-glycosylation and elongation glycosyltransferases were has-miR-1-3p and has-miR-16-5p, respectively. Our findings provide an overview of glycosylation pathways affected by AD and potential regulators of glycosyltransferase expression that deserve further validation and suggest that glycosylation changes occurring in the brains of AD dementia individuals are highly pathway-specific and unique to AD.

Glycomic, Glycoproteomic, and Proteomic Profiling of Philippine Lung Cancer and Peritumoral Tissues: Case Series Study of Patients Stages I-III.

Lung cancer is the leading cause of cancer death and non-small cell lung carcinoma (NSCLC) accounting for majority of lung cancers. Thus, it is important to find potential biomarkers, such as glycans and glycoproteins, which can be used as diagnostic tools against NSCLC. Here, the N-glycome, proteome, and N-glycosylation distribution maps of tumor and peritumoral tissues of Filipino lung cancer patients (n = 5) were characterized. We present several case studies with varying stages of cancer development (I-III), mutation status (EGFR, ALK), and biomarker expression based on a three-gene panel (CD133, KRT19, and MUC1). Although the profiles of each patient were unique, specific trends arose that correlated with the role of aberrant glycosylation in cancer progression. Specifically, we observed a general increase in the relative abundance of high-mannose and sialofucosylated N-glycans in tumor samples. Analysis of the glycan distribution per glycosite revealed that these sialofucosylated N-glycans were specifically attached to glycoproteins involved in key cellular processes, including metabolism, cell adhesion, and regulatory pathways. Protein expression profiles showed significant enrichment of dysregulated proteins involved in metabolism, adhesion, cell-ECM interactions, and N-linked glycosylation, supporting the protein glycosylation results. The present case series study provides the first demonstration of a multi-platform mass-spectrometric analysis specifically for Filipino lung cancer patients.

Multi-glycomic analysis of spheroid glycocalyx differentiates 2- and 3-dimensional cell models.

A multi-glycomic method for characterizing the glycocalyx was employed to identify the difference between 2-dimensional (2D) and 3-dimensional (3D) culture models with two human colorectal cancer cell lines, HCT116 and HT29. 3D cell cultures are considered more representative of cancer due to their ability to mimic the microenvironment found in tumors. For this reason, they have become an important tool in cancer research. Cell-cell interactions increase in 3D models compared to 2D, indeed significant glycomic changes were observed for each cell line. Analyses included the N-glycome, O-glycome, glycolipidome, glycoproteome, and proteome providing the most extensive characterization of the glycocalyx between 3D and 2D thus far. The different glycoconjugates were affected in different ways. In the N-glycome, the 3D cells increased in high-mannose glycosylation and in core fucosylation. Glycolipids increased in sialylation. Specific glycoproteins were found to increase in the 3D cell, elucidating the pathways that are affected between the two models. The results show large structural and biological changes between the 2 models suggesting that the 2 are indeed very different potentially affecting individual outcomes in the study of diseases.

N-Glycan and Glycopeptide Serum Biomarkers in Philippine Lung Cancer Patients Identified Using Liquid Chromatography-Tandem Mass Spectrometry.

Aberrant glycosylation has been extensively reported in cancer, with fundamental changes in the glycosylation patterns of cell-surface and secreted proteins largely occurring during cancer progression. As such, serum glycan and glycopeptide biomarkers have been discovered using mass spectrometry and proposed for cancer detection. Here, we report for the first time potential serum N-glycan and glycopeptide biomarkers for Philippine lung cancer patients. The N-glycan and glycoprotein profiles of a cohort (n = 26 patients, n = 22 age- and gender-matched) of lung cancer patients were analyzed and compared to identify potential N-glycan and glycopeptide serum biomarkers using nano-QToF-MS/MS and ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry dynamic multiple monitoring methods, respectively. Statistical analyses identified differential N-glycan and glycopeptide abundances. The N-glycans were mostly sialylated and sialofucosylated branched structures. The glycopeptides involved proteins in complement and coagulation cascades (p adj = 6.418 × 10-4), innate immunity (p adj = 6.094 × 10-3), acute inflammatory response (p adj = 6.404 × 10-5), defense response (p adj = 2.082 × 10-4), complement activation pathways (p adj = 1.895 × 10-2), and immunoglobulin-mediated immune response pathways (p adj = 4.818 × 10-2). Biomarker models were constructed using serum N-glycans [area under the curve (AUC) = 0.775; 95% CI: 0.617-0.931] and glycopeptides (AUC = 0.959; 95% CI: 0.85-1.0), with glycopeptides having higher accuracies than N-glycans. The results suggest that in the Philippine lung cancer patient sera, specific N-glycans and site-specific glycans are differentially expressed between cases and controls. This report represents the first serum glycan and glycopeptide biomarkers of Philippine lung cancer patients, further demonstrating the utility of mass spectrometry-based glycomic and glycoproteomic methods.

Regio-Specific N-Glycome and N-Glycoproteome Map of the Elderly Human Brain With and Without Alzheimer's Disease.

The proteins in the cell membrane of the brain are modified by glycans in highly interactive regions. The glycans and glycoproteins are involved in cell-cell interactions that are of fundamental importance to the brain. In this study, the comprehensive N-glycome and N-glycoproteome of the brain were determined in 11 functional brain regions, some of them known to be affected with the progression of Alzheimer's disease. N-glycans throughout the regions were generally highly branched and highly sialofucosylated. Regional variations were also found with regard to the glycan types including high mannose and complex-type structures. Glycoproteomic analysis identified the proteins that differed in glycosylation in the various regions. To obtain the broader representation of glycan compositions, four subjects with two in their 70s and two in their 90s representing two Alzheimer's disease subjects, one hippocampal sclerosis subject, and one subject with no cognitive impairment were analyzed. The four subjects were all glycomically mapped across 11 brain regions. Marked differences in the glycomic and glycoproteomic profiles were observed between the samples.

Supplementation with omega-3 or omega-6 fatty acids attenuates platelet reactivity in postmenopausal women.

Postmenopausal women are at increased risk for a cardiovascular event due to platelet hyperactivity. There is evidence suggesting that ω-3 polyunsaturated fatty acids (PUFAs) and ω-6 PUFAs have cardioprotective effects in these women. However, a mechanistic understanding of how these fatty acids regulate platelet function is unknown. In this study, we supplemented postmenopausal women with fish oil (ω-3 fatty acids) or evening primrose oil (ω-6 fatty acids) and investigated the effects on their platelet activity. The effects of fatty acid supplementation on platelet aggregation, dense granule secretion, and activation of integrin αIIbß3 at basal levels and in response to agonist were tested in postmenopausal women following a supplementation and washout period. Supplementation with fish oil or primrose oil attenuated the thrombin receptor PAR4-induced platelet aggregation. Supplementation with ω-3 or ω-6 fatty acids decreased platelet dense granule secretion and attenuated basal levels of integrin αIIbß3 activation. Interestingly, after the washout period following supplementation with primrose oil, platelet aggregation was similarly attenuated. Additionally, for either treatment, the observed protective effects post-supplementation on platelet dense granule secretion and basal levels of integrin activation were sustained after the washout period, suggesting a long-term shift in platelet reactivity due to fatty acid supplementation. These findings begin to elucidate the underlying mechanistic effects of ω-3 and ω-6 fatty acids on platelet reactivity in postmenopausal women. Hence, this study supports the beneficial effects of fish oil or primrose oil supplementation as a therapeutic intervention to reduce the risk of thrombotic events in postmenopausal women. https://clinicaltrials.gov/ct2/show/NCT02629497.

Quantitative glycoproteomics of high-density lipoproteins.

In this work, we developed a targeted glycoproteomic method to monitor the site-specific glycoprofiles and quantities of the most abundant HDL-associated proteins using Orbitrap LC-MS for (glyco)peptide target discovery and QqQ LC-MS for quantitative analysis. We conducted a pilot study using the workflow to determine whether HDL protein glycoprofiles are altered in healthy human participants in response to dietary glycan supplementation.

Glycosylation alterations in serum of Alzheimer's disease patients show widespread changes in N-glycosylation of proteins related to immune function, inflammation, and lipoprotein metabolism.

Introduction: There is an increased need for the development of novel blood-based biomarkers for early detection, prevention, or intervention in Alzheimer's disease (AD). This study sought to determine whether serum glycopeptide analysis holds potential for identifying novel diagnostics and prognostics of AD. Methods: The study involved 195 participants, including 96 patients with an AD diagnosis and 99 controls with no cognitive deficit. Utilizing a validated analytical mass spectrometry method, we monitored the site-specific glycosylation of 52 serum glycoproteins. Results: Partial least-squares discriminant analysis revealed that changes in overall sialylation and fucosylation of serum glycoproteins may be indicators of an AD disease state. Loss of fucosylation of immunoglobulin G1 (IgG1) and IgG2 was indicative of AD diagnosis. Individual glycopeptide analysis found separation between the AD patients and controls on complement proteins and apolipoprotein B. Discussion: The results of this study suggest that serum glycoprofiling may be a promising approach for biomarker discovery.

Docking and mutagenesis studies lead to improved inhibitor development of ML355 for human platelet 12-lipoxygenase.

Human platelet 12-(S)-Lipoxygenase (12-LOX) is a fatty acid metabolizing oxygenase that plays an important role in platelet activation and cardiometabolic disease. ML355 is a specific 12-LOX inhibitor that has been shown to decrease thrombosis without prolonging hemostasis and protect human pancreatic islets from inflammatory injury. It has an amenable drug-like scaffold with nM potency and encouraging ADME and PK profiles, but its binding mode to the active site of 12-LOX remains unclear. In the current work, we combined computational modeling and experimental mutagenesis to propose a model in which ML355 conforms to the "U" shape of the 12-LOX active site, with the phenyl linker region wrapping around L407. The benzothiazole of ML355 extends into the bottom of the active site cavity, pointing towards residues A417 and V418. However, reducing the active site depth alone did not affect ML355 potency. In order to lower the potency of ML355, the cavity needed to be reduced in both length and width. In addition, H596 appears to position ML355 in the active site through an interaction with the 2-methoxy phenol moiety of ML355. Combined, this binding model suggested that the benzothiazole of ML355 could be enlarged. Therefore, a naphthyl-benzothiazole derivative of ML355, Lox12Slug001, was synthesized and shown to have 7.2-fold greater potency than ML355. This greater potency is proposed to be due to additional van der Waals interactions and pi-pi stacking with F414 and F352. Lox12Slug001 was also shown to be highly selective against 12-LOX relative to the other LOX isozymes and more importantly, it showed activity in rescuing human islets exposed to inflammatory cytokines with comparable potency to ML355. Further studies are currently being pursued to derivatize ML355 in order to optimize the additional space in the active site, while maintaining acceptable drug-like properties.

Examination of Carbohydrate Products in Feces Reveals Potential Biomarkers Distinguishing Exclusive and Nonexclusive Breastfeeding Practices in Infants.

BACKGROUND: The stable isotope deuterium dose-to-mother (DTM) technique to estimate nonbreast milk water intake demonstrates that maternal self-report methods of infant feeding overestimate the true prevalence of exclusively breastfeeding practices. OBJECTIVE: We aimed to determine potential monosaccharide and oligosaccharide markers that distinguish between exclusively breastfed (EBF) versus nonexclusively breastfed (non-EBF) infants utilizing LC-MS-based methods. METHODS: Data for the analysis were collected as part of a larger, longitudinal study of 192 breastfed Indonesian infants aged 2 mo and followed up at 5 mo. Feces samples were collected from infants aged 2 mo (n = 188) and 5 mo (n = 184). EBF and non-EBF strata at each time point were determined via the DTM technique. Feces samples were analyzed to determine monosaccharide content using ultra-high-performance LC-triple quadrupole MS (UHPLC-QqQ MS). Relative abundances of fecal oligosaccharides were determined using nano-LC-Chip-quadrupole time-of-flight MS (nano-LC-Chip-Q-ToF MS). RESULTS: At age 2 mo, monosaccharide analysis showed the abundance of fructose and mannose were significantly higher (+377% and +388%, respectively) in non-EBF compared with EBF infants (P <0.0001). Fructose and mannose also showed good discrimination with areas under the curve (AUC) of 0.86 and 0.82, respectively. Oligosaccharide analysis showed that a 6-hexose (Hex6) isomer had good discrimination (AUC = 0.80) between EBF and non-EBF groups at 5 mo. CONCLUSION: Carbohydrate products, particularly fecal mono- and oligosaccharides, differed between EBF and non-EBF infants aged under 6 mo and can be used as potential biomarkers to distinguish EBF versus non-EBF feeding practices.

Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 ± 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a π-stacking interaction with a double bond of arachidonic acid (Δ11), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 ± 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-25418]. However, we were able to increase 15-HpETE to 46 ± 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 ± 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences.

5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1.

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 µM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 µM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 µM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 µM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].