Nano-electrospray Ionization Quadrupole Research Articles

Molecular weight determination of adeno-associate virus serotype 8 virus-like particle either carrying or lacking genome via native nES gas-phase electrophoretic molecular mobility analysis and nESI QRTOF mass spectrometry.

Virus‐like particles (VLPs) are proteinaceous shells derived from viruses lacking any viral genomic material. Adeno‐associated virus (AAV) is a non‐enveloped icosahedral virus used as VLP delivery system in gene therapy (GT). Its success as vehicle for GT is due to its selective tropism, high level of transduction, and low immunogenicity. In this study, two preparations of AAV serotype 8 (AAV8) VLPs either carrying or lacking completely genomic cargo (i.e., non‐viral ssDNA) have been investigated by means of a native nano‐electrospray gas‐phase electrophoretic mobility molecular analyzer (GEMMA) (native nES GEMMA) and native nano‐electrospray ionization quadrupole reflectron time‐of‐flight mass spectrometry (MS) (native nESI QRTOF MS). nES GEMMA is based on electrophoretic mobility principles: single‐charge nanoparticles (NPs), that is, AAV8 particle, are separated in a laminar sheath flow of dry, particle‐free air and a tunable orthogonal electric field. Thus, the electrophoretic mobility diameter (EMD) of a bio‐NP (i.e., diameter of globular nano‐objects) is obtained at atmospheric pressure, which can be converted into its MW based on a correlation. First is the native nESI QRTOF. MS's goal is to keep the native biological conformation of an analyte during the passage into the vacuum. Subsequently, highly accurate MW values are obtained from multiple‐charged species after deconvolution. However, once applied to the analysis of megadalton species, native MS is challenging and requires customized instrumental modifications not readily available on standard devices. Hence, the analysis of AAV8 VLPs via native MS in our hands did not produce a defined charge state assignment, that is, charge deconvolution for exact MW determination was not possible. Nonetheless, the method we present is capable to estimate the MW of VLPs by combining the results from native nES GEMMA and native ESI QRTOF MS. In detail, our findings show a MW of 3.7 and 5.0 MDa for AAV8 VLPs either lacking or carrying an engineered genome, respectively.

Sphingolipid Δ4-desaturation is an important metabolic step for glycosylceramide formation in Physcomitrium patens.

Glycosylceramides are abundant membrane components in vascular plants and are associated with cell differentiation, organogenesis, and protein secretion. Long-chain base (LCB) Δ4-desaturation is an important structural feature for metabolic channeling of sphingolipids into glycosylceramide formation in plants and fungi. In Arabidopsis thaliana, LCB Δ4-unsaturated glycosylceramides are restricted to pollen and floral tissue, indicating that LCB Δ4-desaturation has a less important overall physiological role in A. thaliana. In the bryophyte Physcomitrium patens, LCB Δ4-desaturation is a feature of the most abundant glycosylceramides of the gametophyte generation. Metabolic changes in the P. patens null mutants for the sphingolipid Δ4-desaturase (PpSD4D) and the glycosylceramide synthase (PpGCS), sd4d-1 and gcs-1, were determined by ultra-performance liquid chromatography coupled with nanoelectrospray ionization and triple quadrupole tandem mass spectrometry analysis. sd4d-1 plants lacked unsaturated LCBs and the most abundant glycosylceramides. gcs-1 plants lacked all glycosylceramides and accumulated hydroxyceramides. While sd4d-1 plants mostly resembled wild-type plants, gcs-1 mutants were impaired in growth and development. These results indicate that LCB Δ4-desaturation is a prerequisite for the formation of the most abundant glycosylceramides in P. patens. However, loss of unsaturated LCBs does not affect plant viability, while blockage of glycosylceramide synthesis in gcs-1 plants causes severe plant growth and development defects.

Optimizing sequence coverage for a moderate mass protein in nano-electrospray ionization quadrupole time-of-flight mass spectrometry

Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI–Q-TOF–MS). Use of the final method with trypsin, Lys-C, and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). The sample pretreatment/nESI–Q-TOF–MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA.

Jack bean -mannosidase: Amino acid sequencing and N-glycosylation analysis of a valuable glycomics tool

Jack bean (Canavalia ensiformis) seeds contain several biologically important proteins among which α-mannosidase (EC 3.2.1.24) has been purified, its biochemical properties studied and widely used in glycan analysis. In the present study, we have used the purified enzyme and derived its amino acid sequence covering both the known subunits (molecular mass of ∼66,000 and ∼44,000 Da) hitherto not known in its entirety. Peptide de novo sequencing and structural elucidation of N-glycopeptides obtained either directly from proteolytic digestion or after zwitterionic hydrophilic interaction liquid chromatography solid phase extraction-based separation were performed by use of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry and low-energy collision-induced dissociation experiments. De novo sequencing provided new insights into the disulfide linkage organization, intersection of subunits and complete N-glycan structures along with site specificities. The primary sequence suggests that the enzyme belongs to glycosyl hydrolase family 38 and the N-glycan sequence analysis revealed high-mannose oligosaccharides, which were found to be heterogeneous with varying number of hexoses viz, Man8-9GlcNAc2 and Glc1Man9GlcNAc2 in an evolutionarily conserved N-glycosylation site. This site with two proximal cysteines is present in all the acidic α-mannosidases reported so far in eukaryotes. Further, a truncated paucimannose type was identified to be lacking terminal two mannose, Man1(Xyl)GlcNAc2 (Fuc).

Ionization suppression effect of phosphopeptides in nano-electrospray ionization quadrupole time-of-flight mass spectrometry

Protein phosphorylation is an important post-translational modification (PTM), and involves in many cell activities and disease processes. Nowadays, mass spectrometry (MS) is a powerful tool in peptide-based proteomics. But phosphopeptides, especially multiply phosphorylated peptides, are difficult to be detected by MS. One opinion is that the presence of unphosphorylated peptides lead to the ionization suppression of phosphopeptides. But contrary opinion exists. Moreover, the suppression effect caused by different types of phosphopeptides hasn't been studied yet. In this study, a set of synthetic standard phosphopeptides (singly, doubly and triply phosphorylated peptides) with the same amino acid sequence, and the unphosphorylated peptides were injected into mass spectrometer with or without mixing. Through the ratios of signal intensities of the phosphopeptides before and after mixing, we confirmed that the signals of phosphopeptides were suppressed by those of the unphosphorylated peptides. In addition, the signals of the doubly phosphorylated peptides were slightly suppressed by those of the singly phosphorylated peptides with the same amino acid sequence. The signals of the triply phosphorylated peptides were notably suppressed by the singly and doubly phosphorylated peptides with the same amino acid sequence. Therefore, the singly and multiply phosphorylated peptides should be fractionated and enriched before mass spectrometry analysis.

Highly efficient microscale purification of glycerophospholipids by microfluidic cell lysis and lipid extraction for lipidomics profiling.

This article presents a novel method for small-scale lipidomics of bacterial cells by integrating extraction of glycerophospholipids on a microchip with a nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometer (nanoESI-Q-TOF MS/MS). The standard starting point for typical macroscale lipid analysis is a multiphase liquid-liquid extraction. Working with small populations of cells (1 to about 1000) requires a scaled down process in order to minimize dilution and facilitate the interface with microscale separation methods for sample cleanup and introduction to mass spectrometry. We have developed a microfluidic system that allows for lysis of bacterial cells, capture of lipids, and elution of captured lipids from a solid phase for microscale purification of lipids. The best on-chip extraction efficiency for glycerophospholipids was as high as 83.3% by integrating silica beads as the packing material with methanol as the eluent. A total of 10 successive measurements were evaluated indicating that the microchip packed with fresh silica beads is capable of being reused four times without any loss in the lipid extraction process. The initial screening based on high-resolution tandem mass spectrometry data along with a discovery profiling approach revealed the presence of 173 identified phospholipid species from microfluidic cell extracts. This work demonstrates the potential of incorporating microchip-based lipid extraction into cellular lipidomics research.

On the structural diversity of Shiga toxin glycosphingolipid receptors in lymphoid and myeloid cells determined by nanoelectrospray ionization tandem mass spectrometry

Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Galalpha4Galbeta4Glcbeta1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), respectively, which represent the targets of Stxs on many different cell types. B-cell-derived Raji cells and THP-1 cells of monocytic origin are widely used for the investigation of Stx-mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP-1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) with collision-induced dissociation (CID) in conjunction with Stx1 as well as anti-Gb3Cer and anti-Gb4Cer antibodies. Using the combination of a thin-layer chromatography (TLC) overlay assay and MS(1) and MS(2) analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1-receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP-1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP-1 cells we observed the expression of Gb4Cer in THP-1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short- and long-chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx-susceptible cells.

Speciation of manganese binding to biomolecules in pine nuts (Pinus pinea) by two‐dimensional liquid chromatography coupled to ultraviolet and inductively coupled plasma mass spectrometry detectors followed by identification by electrospray ionization mass spectrometry

Advances in analytical methodology for speciation of manganese in pine nuts are presented in this work. The approach is based on the use of orthogonal chromatographic systems, namely size-exclusion chromatography (SEC) of the extracts and strong anion exchange (IEC) of the fractions collected by the first column. In both columns, manganese elution is first monitored by a quadrupole inductively coupled plasma mass spectrometry (ICP-MS) instrument equipped with an octopole reaction cell and an ultraviolet (UV) detector. SEC is performed by using two columns covering the molecular weight range from <10 to 70 kDa that allows an initial screening of the molecular weight of the Mn species. The higher resolution capability of the low molecular weight range column is the reason to use the latter for further experiments. The fraction from SEC-ICP-MS in which Mn is present at highest concentration is submitted to IEC-ICP-MS allowing Mn-citrate and MnCl(2) identification by retention time matching with standards. The concentration of these species is estimated to be 75 and 125 microg kg(-1) (as Mn), respectively, in the pine nuts samples and the presence of Mn-citrate is confirmed by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nESI-QqTOF-MS). In the same fraction, a third Mn-containing peak is detected in the IEC-UV-ICP-MS chromatogram. This peak corresponds to a protein containing Mn that was later submitted to a tryptic digestion and analyzed by nESI-QqTOF. The MS/MS data of a doubly charged peptide are used to obtain the sequence of the protein with the Mascot search engine. The peak turned out to be isocitrate dehydrogenase, a protein commonly associated with Mn.

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

Glycosphingolipids in vascular endothelial cells: relationship of heterogeneity in Gb3Cer/CD77 receptor expression with differential Shiga toxin 1 cytotoxicity

Shiga toxin (Stx) 1 binds to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) and injures human endothelial cells. In order to gain insight into Stx1-induced cellular impairment, we analysed in detail the molecular heterogeneity of Stx1 receptors in two endothelial cell lines differing in their Stx1-sensitivity. We observed a moderate sensitivity to Stx1 of human brain microvascular endothelial cells (HBMECs, CD(50) > 200 ng/ml), but a considerably higher mortality rate in cultures of EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (CD(50) of 0.2 ng/ml). Immunofluorescence microscopy demonstrated the presence of Gb3Cer in both cell lines, but showed an enhanced content of Gb3Cer in EA.hy 926 cells. Solid phase overlay binding assays of isolated GSLs combined with nanoelectrospray ionization quadrupole time-of-flight mass spectrometry demonstrated a balanced proportion of Gb3Cer and globotetraosylceramide (Gb4Cer) in HBMECs, but an increase of Gb3Cer and absence of Gb4Cer in EA.hy 926 cells. Gb3Cer species with C24:1/C24:0 fatty acids were found to dominate over those with C16:0 fatty acids in EA.hy 926 cells, but were similarly distributed in HBMECs. Reverse transcriptase polymerase chain reaction indicated the concomitant presence of Gb3Cer and Gb4Cer synthases in HBMECs, whereas EA.hy 926 cells expressed Gb3Cer synthase, but completely lacked Gb4Cer synthase. This deficiency, resulting in the accumulation of Gb3Cer in EA.hy 926 cells, represents the most prominent molecular reason that underlies the different Stx1 sensitivities of HBMECs and EA.hy 926 endothelial cells.

Alternative Neisseria spp. type IV pilin glycosylation with a glyceramido acetamido trideoxyhexose residue

The importance of protein glycosylation in the interaction of pathogenic bacteria with their host is becoming increasingly clear. Neisseria meningitidis, the etiological agent of cerebrospinal meningitis, crosses cellular barriers after adhering to host cells through type IV pili. Pilin glycosylation genes (pgl) are responsible for the glycosylation of PilE, the major subunit of type IV pili, with the 2,4-diacetamido-2,4,6-trideoxyhexose residue. Nearly half of the clinical isolates, however, display an insertion in the pglBCD operon, which is anticipated to lead to a different, unidentified glycosylation. Here the structure of pilin glycosylation was determined in such a strain by "top-down" MS approaches. MALDI-TOF, nanoelectrospray ionization Fourier transform ion cyclotron resonance, and nanoelectrospray ionization quadrupole TOF MS analysis of purified pili preparations originating from N. meningitidis strains, either wild type or deficient for pilin glycosylation, revealed a glycan mass inconsistent with 2,4-diacetamido-2,4,6-trideoxyhexose or any sugar in the databases. This unusual modification was determined by in-source dissociation of the sugar from the protein followed by tandem MS analysis with collision-induced fragmentation to be a hexose modified with a glyceramido and an acetamido group. We further show genetically that the nature of the sugar present on the pilin is determined by the carboxyl-terminal region of the pglB gene modified by the insertion in the pglBCD locus. We thus report a previously undiscovered monosaccharide involved in posttranslational modification of type IV pilin subunits by a MS-based approach and determine the molecular basis of its biosynthesis.

Mass spectrometric detection of CP4 EPSPS in genetically modified soya and maize

The potential of protein fractionation hyphenated to mass spectrometry (MS) to detect and characterize the transgenic protein present in Roundup Ready soya and maize has been investigated. Genetically modified (GM) soya and maize contain the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4, which confers resistance to the herbicide glyphosate. The GM soya and maize proteomes were fractionated by gel filtration, anion-exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) prior to MS. This facilitated detection of a tryptic peptide map of CP4 EPSPS by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nanoelectrospray ionization quadrupole time-of-flight (nanoESI-QTOF) MS. Subsequently, sequence information from the CP4 EPSPS tryptic peptides was obtained by nanoESI-QTOF MS/MS. The identification was accomplished in 0.9% GM soya seeds, which is the current EU threshold for food-labeling requirements.

Coliphage N4 N-Acetylmuramidase Defines a New Family of Murein Hydrolases

Escherichia coli phage N4 infection leads to delayed host cell lysis, 3000 particles per infected bacterium and a small plaque phenotype. We show that bacteriophage N4 encodes a murein hydrolase (gp61) that is essential for N4 plaque-forming ability. gp61 has a high level of sequence similarity to hypothetical proteobacterial proteins, and Vibrio harveyi phage VHML ORF 19. Nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS) analysis of muropeptides from purified gp61 digestion of E. coli peptidoglycan indicates that gp61 is an N-acetylmuramidase. The EGGY motif present near the N terminus of gp61 and its homologs contains the glutamic acid residue essential for enzymatic activity. These results provide evidence that N4 gp61 and its homologs define a new family of N-acetylmuramidases (pfam05838.4, DUF847, COG3926). In contrast to its homologs, gp61 contains an N-terminal signal sequence. When expressed at levels present during phage infection, gp61 localizes primarily to the cell inner membrane; in contrast, over-expression of recombinant N4 gp61 is sufficient for rapid cell lysis. Overproduction of the recombinant Salmonella typhimurium (STM0016) homolog is sufficient for cell lysis only when fused to the gp61 N-terminal signal sequence. The results of subcellular localization and of mutagenesis of the gp61 N-terminal signal sequence indicate that gp61 must be released from the inner membrane to be catalytically active.

Analysis of Oversulfation in a Chondroitin Sulfate Oligosaccharide Fraction from Bovine Aorta by Nanoelectrospray Ionization Quadrupole Time-of-Flight and Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry

A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B- and C-type ions and their respective complementary Y- and Z-type fragment ions.

Identification of Recombinant Human Parathyroid Hormone (1–34) by Nanoelectrospray Ionization-Quadrupole Time-of-Flight Tandem Mass Spectrometry

Recombinant human PTH (1–34) was identified by electrospray ionization-quadrupole time-of-flight tandem mass spectrometry (ESI-TOF-MS). The calculated molecular weight of PTH (1–34) was 4117.76 by ESI-MS, and the measured value was 4115.21. The percent of measurement error was only 0.06%. PTH (1–34) was digested by trypsin at 37° overnight. The tryptic-digested peptides were analyzed, and then four charged peptides were selected to determine their amino acid sequence by ESI-MS/MS. Through analysis of the a, b and y series ions, the sequence of peptide m/z 728.4 determined by ESI-MS/MS was VSEIQLMHNLGK, and the database search results of the other three peptides showed that both belonged to PTH (1–34). The study proved that the primary structure of the recombinant PTH (1–34) was accurate, and there was no amino acid deletion, mutation, or modification in its expression and in the purification process of PTH. It also showed that nanoESI-MS/MS was a good method to identify protein with the advantages of high sensitivity, fast analysis, and accuracy.

Electrospray ionization quadrupole time‐of‐flight tandem mass spectrometric analysis of hexamethylenediamine‐modified maltodextrin and dextran

A combined methodology for obtaining at the preparative scale and characterization by nanoelectrospray ionization (nanoESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and tandem MS (MS/MS) of linear polysaccharides modified at the reducing end is presented. Two polydisperse maltodextrins (1000 and 3000 Da) and a high molecular weight polydisperse dextran (6000 Da) were coupled with hexamethylenediamine (HMD). The coupling products were analyzed by nanoESI-QTOF-MS in the positive ion mode and MS/MS using collision-induced dissociation (CID) at low energies. In the HMD-M1000 mixture, the polysaccharide chains containing from 2 to 8 Glc residues were detected, while in HMD-M3000 we identified a complete series of chains containing from 8 to 21 Glc moieties. The employed ESI conditions enhanced the detection of chains with up to 46 Glc residues in the HMD-D6000 sample. By optimized MS/MS, HMD-modified polysaccharides of 3, 4, 5, 12 and 46 degrees of polymerization yielded product ion spectra exhibiting the whole set of Y- and B-fragment ions. The MS structural data were obtained within a few minutes of signal acquisition, with a sample consumption situating the analysis sensitivity in the picomolar range.

Characterization of Intact Hemoglobin and Oxaliplatin Interaction by Nanoelectrospray Ionization Tandem Mass Spectrometry

Mass spectrometric (MS) detection of intact hemoglobin (Hb) adducts presents considerable analytical challenges because of the noncovalent association of the 4 subunits of Hb, and MS characterization of the interaction of intact Hb with platinum drugs has not been reported. We developed a technique for detecting intact Hb and its drug adduct and studied the interactions between intact Hb and oxaliplatin. We incubated a series of mixtures of Hb and oxaliplatin at 37 degrees C for 24 h or 5 days to investigate adduct formation. Blood samples from colorectal cancer patients undergoing oxaliplatin treatment were analyzed for novel adducts of intact Hb-oxaliplatin, which were characterized with nanoelectrospray ionization quadrupole time-of-flight MS. Two intact Hb adducts, one with the whole oxaliplatin molecule and the other with oxaliplatin losing the oxalate ligand, were identified. Analysis of erythrocytes from the cancer patients provided direct evidence that oxaliplatin accumulated as Hb adducts in erythrocytes. A higher fraction (approximately 70%) of Hb was bound to oxaliplatin in erythrocytes from a patient who could not tolerate oxaliplatin treatment than in erythrocytes from another patient who benefited from this treatment. The nanoelectrospray tandem MS technique enabled determination of the intact Hb tetramer and its association with oxaliplatin. Hb-oxaliplatin adducts in erythrocytes may serve as a clinical biomarker for toxic response and treatment efficacy.

Application of combined high‐performance thin‐layer chromatography immunostaining and nanoelectrospray ionization quadrupole time‐of‐flight tandem mass spectrometry to the structural characterization of high‐ and low‐affinity binding ligands of Shiga toxin 1

Shiga toxin 1 (Stx1) represents an AB5 toxin produced by enterohemorrhagic Escherichia coli, which cause gastrointestinal diseases in humans that are often followed by potentially fatal systemic complications, such as acute encephalopathy and hemolytic uremic syndrome. The expression of the preferential Stx1 receptor, Gb3Cer/CD77 (Gal alpha1-4Gal beta1-4Glc beta1-1Cer), is one of the primary determinants of susceptibility to tissue injury. Due to the clinical importance of this life-threatening toxin, a combined strategy of preparative high-performance thin-layer chromatography (HPTLC) overlay assay and mass spectrometry was developed for the detection and structural characterization of Stx1-binding glycosphingolipids (GSLs). A preparation of neutral GSLs from human erythrocytes, comprising 21.4% and 59.1% of the high- and low-affinity Stx1-binding ligands Gb3Cer/CD77 and Gb4Cer, respectively, was separated on silica gel precoated HPTLC plates and probed for the presence of Stx1 receptors. Stx1 positive on the one hand and anti-Gb3Cer/CD77 and anti-Gb4Cer antibody positive bands from parallel reference runs on the other hand were extracted with chloroform/methanol/water (30/60/8, v/v/v). These crude extracts were used without any further purification for a detailed structural analysis by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) in the negative ion mode. In all extracts investigated, neutral GSLs were detected as singly charged deprotonated molecular ions, [M-H]-, and neither buffer-derived salt adducts nor coextracted contaminants from the overlay assay procedure or the silica gel layer were observed. For the structural characterization of Stx1- and antibody-binding GSLs low-energy collision-induced dissociation (CID) was applied to high and low abundant receptor species of the crude extracts. All MS/MS spectra obtained contained full series of Y-type ions, B-type ions and additional ions generated by ring cleavages of the sugar moiety. Only analytical quantities in the microgram scale of a single GSL species within the complex GSL mixture were required for the structural MS characterization of Stx1 ligands as Gb3Cer/CD77 and Gb4Cer. This effective combined HPTLC/MS procedure offers a broad range of applications, not only for toxins of bacterial origin, but also for any GSL-binding agents such as plant-derived lectins or human proteins with yet unknown binding specificities.

Intact human holo‐transferrin interaction with oxaliplatin

We report the interaction of intact human holo-transferrin (holo-Tf) with oxaliplatin (an anticancer drug), and the characterization of a complex composed of (1:1) intact holo-Tf and the parent oxaliplatin molecule using nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS). The molecular weight of this complex was determined to be 80,077 Da, which was an increase of 397 mass units compared to the protein alone (79,680 Da), suggesting that a parent drug molecule was bound to the intact protein. We further examined the interaction between the intact protein and oxaliplatin using size-exclusion high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS). The protein complex and free oxaliplatin were separated by HPLC and quantitatively determined by simultaneous monitoring of both 195Pt and 56Fe using ICPMS. The HPLC/ICPMS detected both Pt and Fe signals at retention time of 2.6 min, identifying the protein-drug complex. The Fe signal at 2.6 min did not change with the increase in incubation time of the reaction mixture containing holo-Tf and oxaliplatin, while the Pt signal at the same retention time increased over time, further demonstrating that the formation of this complex does not affect the protein-bound Fe. The binding constant of the (1:1) intact human holo-Tf-oxaliplatin complex was determined to be 7.7x10(5) M-1. Both nanoESI-MS and HPLC/ICPMS results support that the holo-Tf and parent oxaliplatin molecules form complexes through non-covalent binding, suggesting that holo-Tf may be a useful carrier for oxaliplatin delivery.

FIRST EVIDENCE OF PALYTOXIN ANALOGUES FROM AN OSTREOPSIS MASCARENENSIS (DINOPHYCEAE) BENTHIC BLOOM IN SOUTHWESTERN INDIAN OCEAN1

Benthic dinoflagellates of the genus Ostreopsis Schmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom of Ostreopsis mascarenensis Quod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology of O. mascarenensis was studied by LM and SEM. Preliminary screening of a crude extract of an O. mascarenensis bloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol‐soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin‐A and ‐B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time‐of‐flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ion m/z 327. An on‐line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom of O. mascarenensis. These results suggest that O. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area.