Hydrophilic Interaction Liquid Chromatography-mass Spectrometry Research Articles

Characterization of heparin oligosaccharides binding specifically to antithrombin III using mass spectrometry.

Heparan sulfate (HS) is a sulfated glycosaminoglycan attached to a core protein on the cell surface. Protein binding to cell surface HS is a key regulatory event for many cellular processes such as blood coagulation, cell proliferation, and migration. The concept whereby protein binding to HS is not random but requires a limited number of sulfation patterns is becoming clear. Here we describe a hydrophobic trapping assay for screening a library of heparin hexasaccharides for binders to antithrombin III (ATIII). The hexasaccharide compositions are defined with their building block content in the following format: (DeltaHexA:HexA:GlcN:SO 3:Ac). Of five initial compositions present in the library, (1:2:3:6:1), (1:2:3:7:1), (1:2:3:7:0), (1:2:3:8:0), and (1:2:3:9:0), only two are shown to bind ATIII, namely, (1:2:3:8:0) and (1:2:3:9:0). The use of amide hydrophilic interaction (HILIC) liquid chromatography-mass spectrometry permitted reproducible quantitative analysis of the composition of the initial library as well as that of the binding fraction. The specificity of the hexasaccharides binding ATIII was confirmed by assaying their ability to enhance ATIII-mediated inhibition of Factor Xa in vitro.

Targeted Metabolomics Analysis of Campylobacter coli VC167 Reveals Legionaminic Acid Derivatives as Novel Flagellar Glycans

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.

Functional Characterization of the Flagellar Glycosylation Locus in Campylobacter jejuni 81–176 Using a Focused Metabolomics Approach

Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81-176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81-176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-L-glycero-alpha-L-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways.

A 96-well single-pot liquid–liquid extraction, hydrophilic interaction liquid chromatography–mass spectrometry method for the determination of muraglitazar in human plasma

A single-pot liquid–liquid extraction (LLE) with hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC/MS/MS) method has been developed and validated for the determination of muraglitazar, a hydrophobic diabetes drug, in human plasma. To 0.050 ml of each plasma sample in a 96-well plate, the internal standard solution in acetonitrile and toluene were added to extract the compound of interest. The plate was vortexed, followed by centrifugation. The organic layer was then directly injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Thermohypersil_Keystone, Hypersil silica column (3 mm × 50 mm, 3 μm). The mobile phase contained 85% of methyl t-butyl ether and 15% of 90/10 (v/v) acetonitrile/water with 0.3% trifluoroacetic acid. Post-column mobile phase of 50/50 (v/v) acetonitrile/water containing 0.1% formic acid was added. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API 4000. The standard curve, ranged from 1 to 1000 ng/ml, was fitted to a 1/ x weighted quadratic regression model. This single-pot LLE approach effectively eliminated time-consuming organic layer transfer, dry-down, and sample reconstitution steps, which are essential for a conventional liquid–liquid extraction procedure. The modified mobile phase was more compatible with the direct injection of the commonly used extraction solvents in LLE. Furthermore, the modified mobile phase improved the retention of muraglitazar, a hydrophobic compound, on the normal phase silica column. The validation results demonstrated that this method was rugged and suitable for analyzing muraglitazar in human plasma. In comparison with a revised-phase LC/MS/MS method, this single-pot LLE, HILIC/MS/MS method improved the detection sensitivity by more than four-fold based upon the LLOQ signal to noise ratio. This approach may be applied to other hydrophobic compounds with proper modification of the mobile phase compositions.

Investigation of the toxin profile of Greek mussels Mytilus galloprovincialis by liquid chromatography—mass spectrometry

Samples of Mytilus galloprovincialis were harvested from five different locations in Thermaikos gulf, Greece after harmful algae bloom. All of the mussel samples were found positive by mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins. Liquid chromatography (LC) coupled with mass spectrometry (MS) was used to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and yessotoxins (YTXs). In order to investigate the presence of okadaic acid esters, alkaline hydrolysis was performed for all the samples, and LC–MS analyses were carried out on the samples before and after hydrolysis. Hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS) analyses were also carried out to investigate the presence of domoic acid and paralytic shellfish poisoning (PSP) toxins at trace levels. All of the samples were found to be contaminated only with okadaic acid at levels 0.10–0.20 μg/g.

Hydrophilic interaction liquid chromatography–mass spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins

Hydrophilic interaction liquid chromatography (HILIC) was examined for the separation of paralytic shellfish poisoning (PSP) toxins using the stationary phase TSK-gel Amide-80 ®. The parameters tested included type of organic modifier and percentage in the mobile phase, buffer concentration, pH, flow rate and column temperature. Using mass spectrometric (MS) detection, the HILIC column allowed the determination of all the major PSP toxins in one 30 min analysis with a high degree of selectivity and sensitivity. The high percentage of organic modifier in the mobile phase and the omission of ion pairing reagents, both favored in HILIC, provided limits of detection (LOD) in the range 50–1000 nM in selected ion monitoring (SIM) mode on a single quadrupole LC–MS system. LOD in selected reaction monitoring (SRM) mode on a sensitive triple quadrupole system were as low as 5–30 nM. Excellent linearity of response was observed.

Analysis of cyanobacterial toxins by hydrophilic interaction liquid chromatography–mass spectrometry

The combination of hydrophilic interaction liquid chromatography with electrospray mass spectrometry (HILIC–MS) has been investigated as a tool for the analysis of assorted toxins produced by cyanobacteria. Toxins examined included saxitoxin and its various analogues ( 1– 18), anatoxin-a (ATX-a, 19), cylindrospermopsin (CYN, 20), deoxycylindrospermopsin (doCYN, 21), and microcystins-LR ( 22) and -RR ( 23). The saxitoxins could be unequivocally detected in one isocratic analysis using a TSK gel Amide-80 column eluted with 65% B, where eluent A is water and B is a 95% acetonitrile/water solution, both containing 2.0 mM ammonium formate and 3.6 mM formic acid. The analysis of ATX-a, CYN and doCYN required 75% B isocratic. Simultaneous determination of 1– 21 was also possible by using gradient elution. HILIC proved to be suitable for the analysis of microcystins, but peak shape was not symmetric and it was concluded that these compounds are best analysed using existing reversed-phase methods. The HILIC–MS method was applied to the analysis of field and cultured samples of Anabaena circinalis and Cylindrospermopsis raciborskii. In general, the method proved quite robust with similar results obtained in two different laboratories using different instrumentation.

Toxic Tides

Research ArticleOpen AccessToxic tides. J Tibbetts J Tibbetts Search for more papers by this author Published:1 July 1998https://doi.org/10.1289/ehp.98106a326Cited by:11AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit FiguresReferencesRelatedDetailsCited by Morabito S, Silvestro S and Faggio C (2017) How the marine biotoxins affect human health, Natural Product Research, 10.1080/14786419.2017.1329734, 32:6, (621-631), Online publication date: 19-Mar-2018. Van Wagoner R, Satake M and Wright J (2014) Polyketide biosynthesis in dinoflagellates: what makes it different?, Natural Product Reports, 10.1039/C4NP00016A, 31:9, (1101) Dell’Aversano C (2011) Hydrophilic Interaction Liquid Chromatography–Mass Spectrometry (HILIC–MS) of Paralytic Shellfish Poisoning Toxins, Domoic Acid, and Assorted Cyanobacterial Toxins Hydrophilic Interaction Liquid Chromatography (HILIC) and Advanced Applications, 10.1201/b10609-7, (105-132), Online publication date: 17-Feb-2011. Ciminiello P, Dell'Aversano C, Fattorusso E, Forino M, Tartaglione L, Boschetti L, Rubini S, Cangini M, Pigozzi S and Poletti R (2010) Complex toxin profile of Mytilus galloprovincialis from the Adriatic sea revealed by LC–MS, Toxicon, 10.1016/j.toxicon.2009.07.037, 55:2-3, (280-288), Online publication date: 1-Feb-2010. Still K and Mohapatra A (2009) Biotoxins Information Resources in Toxicology, 10.1016/B978-0-12-373593-5.00010-0, (91-102), . Ciminiello P, Dell' Aversano C, Fattorusso E and Forino M (2009) Chapter 1 Recent Developments in Mediterranean Harmful Algal Events , 10.1016/S1872-0854(09)00001-0, (1-41), . Mao H, Thorne J, Pharr J and Gawley R (2006) Effect of crown ether ring size on binding and fluorescence response to saxitoxin in anthracylmethyl monoazacrown ether chemosensors, Canadian Journal of Chemistry, 10.1139/v06-093, 84:10, (1273-1279), Online publication date: 1-Oct-2006. Guy R (2005) Red Tide Encyclopedia of Toxicology, 10.1016/B0-12-369400-0/00835-8, (628-630), . Kirkpatrick B, Fleming L, Squicciarini D, Backer L, Clark R, Abraham W, Benson J, Cheng Y, Johnson D, Pierce R, Zaias J, Bossart G and Baden D (2004) Literature review of Florida red tide: implications for human health effects, Harmful Algae, 10.1016/j.hal.2003.08.005, 3:2, (99-115), Online publication date: 1-Apr-2004. Hales S, Weinstein P and Woodward A (1999) Ciguatera (Fish Poisoning), El Nino, and Pacific Sea Surface Temperatures, Ecosystem Health, 10.1046/j.1526-0992.1999.09903.x, 5:1, (20-25), Online publication date: 1-Mar-1999. Fleming L, Easom J, Baden D, Rowan A and Levfn B (2016) Review Article: Emerging Harmful Algal Blooms and Human Health: Pfiesteria and Related Organisms, Toxicologic Pathology, 10.1177/019262339902700511, 27:5, (573-581), Online publication date: 1-Sep-1999. Vol. 106, No. 7 July 1998Metrics About Article Metrics Publication History Originally published1 July 1998Published in print1 July 1998 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.