Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell‐to‐cell or cell‐to‐protein interactions. In this study, qualitative and quantitative analyses of GSL‐derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL‐derived glycans from the endothelial cells and islets, respectively, were characterized by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and collision‐induced fragmentation using positive‐ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α‐Gal‐terminated GSL was not detected but NeuGc‐terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL‐derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI‐TOF MS. The structural information of the GSL‐derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Automated normal phase nano high performance liquid chromatography/matrix assisted laser desorption/ionization mass spectrometry for analysis of neutral and acidic glycosphingolipids

The coupling of nano high-performance liquid chromatography (nanoHPLC) with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) via an automatic spotting roboter was developed and adapted for the first time for the analysis of complex mixtures of glycosphingolipids (GSLs). The 2,5-dihydroxybenzoic acid and 6-azo-2-thiothymine matrix systems were adjusted to concurrently meet the requirements for reproducible and homogeneous crystal formation with the liquid chromatography (LC) eluent under the variable LC solvent composition over the course gradient and high ionization efficiency of the GSL species, without the need for recrystallization. Precise adjustment of the automatic spotting parameters in terms of matrix flow rate, on-tip collection time of the matrix/LC eluent solution and the matrix spotting mode, i.e., continuous and discontinuous, was accomplished to collect individually nanoHPLC-separated species within distinct spots and consequently recover by MALDI MS screening all major and minor GSL species in the mixtures. The nanoHPLC/MALDI MS coupling protocol was developed and applied to a mixture of neutral GSLs purified from human erythrocytes and a monosialoganglioside mixture expressed by the murine MDAY-D2 cell line. Additionally, on-line nanoHPLC/MALDI doping with lithium cations of individually separated neutral GSLs was introduced to enhance data interpretation of the GSL MS pattern, while preserving the same level of information and ultimately to enhance structural assignment of components of interest. The method is demonstrated to be highly sensitive, reaching the low femtomole level of detection of individual GSL species and is highlighted as a versatile analytical tool for glycolipidomic studies. Figure Automatic LC/MALDI MS profiling of glycosphingolipids Mostafa Zarei and Stephan Kirsch contributed equally to this work.     

Software utilities for the interpretation of mass spectrometric data of glycoconjugates: application to glycosphingolipids of human serum

Glycosphingolipids (GSLs) are major components of the outer leaflet of the cell membrane. These lipids are involved in many cell surface events and show disease‐related expression changes. GSLs could thus serve as useful targets for biomarker discovery. The GSL structure is characterized by two entities: a hydrophilic glycan and a hydrophobic ceramide moiety. Both components exhibit numerous structural variations, the combination of which results in a large diversity of GSL structures that can potentially exist. Mass spectrometry (MS) is a powerful tool for high‐throughput analysis of GSL expression analysis and structural elucidation. Yet, the assignment of GSL structures using MS data is tedious and demands highly specialized expertise. SysBioWare, a software platform developed for MS data evaluation in glycomics, was here applied for the MS analysis of human serum GSLs. The program was tuned to provide automated compositional assignment, supporting a variety of glycan and ceramide structures. Upon in silico fragmentation, the masses of predicted ions arising from cleavages in the glycan as well as the ceramide moiety were calculated, thus enabling structural characterization of both entities. Validation of proposed structures was achieved by matching in silico calculated fragment ions with those of experimental MS/MS data. These results indicate that SysBioWare can facilitate data interpretation and, furthermore, help the user to deal with large sets of data by supporting management of MS and non‐MS data. SysBioWare has the potential to be a powerful tool for high‐throughput glycosphingolipidomics in clinical applications. Copyright © 2010 John Wiley & Sons, Ltd.     

A quick and easy method to identify bacteria by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry

Concerns with water quality have increased in recent years, in part due to the more frequent contamination of water by pathogens like E. coli and L. pneumophila. Current methods for the typing of bacteria in water samples are based on culture of samples on specific media. These techniques are time‐consuming, subject to the impact of interferents and do not totally meet all the requirements of prevention. There is a need for accurate and rapid identification of these microorganisms. This report deals with the detection of bacteria, more precisely of Legionella spp., and the development of an analytical strategy for a rapid and unambiguous identification of these pathogens in water from diverse origins. Therefore, a protein mass mapping using matrix‐assisted laser desorption/ionisation mass spectrometry (MALDI MS) of whole bacteria combined with a home‐made database of bacteria spectra is applied. A large variety of different bacteria and microorganisms is used to approach the actual composition of samples with numerous interferents. The objective is to propose a universal method for sampling preparation before MALDI MS analysis and optimised spectrometric conditions for reproducible intense peaks. Several experimental factors known to influence signal quality such as time and media of culture have been studied. The proposed method gives promising results for a sure differentiation of Legionella species and subspecies and a rapid identification of bacteria which are the most dangerous or difficult to eradicate. This method is easy to perform with an excellent reproducibility. The analytical protocol and the corresponding database were validated on samples from different origins (cooling tower, plumbing hot water). Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Qualification and application of a liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the determination of trastuzumab in rat plasma

An liquid chromatography–quadrupole time‐of‐flight (QqTOF) mass spectrometric method was developed for the determination of humanized or human monoclonal antibodies in rat plasma at the early drug discovery stage. Trastuzumab was used as a model monoclonal antibody. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC‐TOF‐MS/MS analysis of specific signature peptides in the positive ion mode using electrospray ionization for analysis. A stable isotope‐labeled signature peptide was also used as internal standard. A quadratic regression (weighted 1/concentration²), with an equation y = ax² + bx + c, was used to fit calibration curves over the concentration range of 0.500–100 µg/mL for trastuzumab. Samples from a pharmacokinetic study in rat were analyzed by this qualified LC‐TOF‐MS/MS method and concentrations were compared with those generated by enzyme linked immunosorbent assays method. The LC‐TOF‐MS/MS method was accurate and precise, with quantitative results comparable with those of ELISA. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. Within‐run accuracy ranged from 1.53 to 9.20% with precision values ≤10.29%. This LC‐TOF‐MS/MS method approach could be used as a complementary method for humanized or human monoclonal antibodies at the early drug discovery stage. Copyright © 2015 John Wiley & Sons, Ltd.     

Detection of pathogenic Verticillium spp. using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Verticillium spp. have been listed by the European and Mediterranean Plant Protection Organization (EPPO) and China as plant quarantine pests. Although attempts have been made to develop a simple routine laboratory assay to detect these organisms, none are routinely used. We describe for the first time a robust assay for reliable identification of Verticillium spp. using protein fingerprinting data obtained by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry(MALDI‐TOF‐MS). Several sample preparation methods and matrices were investigated to improve mass spectra for the routine identification of six species of Verticillium spp.(Verticillium dahiliae, V. alboatrum, V. fungicola, V. nigrescens, and V. lecanii) by MALDI‐TOF‐MS. Using the optimized experimental method, we constructed a protein fingerprint database for six species of Verticillium and established a analysis criteria of log(Score). This MALDI‐TOF‐MS protocol should prove useful as a rapid and reliable assay for distinguishing different Verticillium spp. Copyright © 2009 John Wiley & Sons, Ltd.     

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 Galα4Galβ4Glcβ1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAcβ3Galα4Galβ4Glcβ1Cer), 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 MS¹ and MS² 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. Copyright © 2010 John Wiley & Sons, Ltd.     

Enhanced matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis of bacteriophage major capsid proteins with β‐mercaptoethanol pretreatment

Bacteriophage (phage) proteins have been analyzed previously with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). However, analysis of phage major capsid proteins (MCPs) has been limited by the ability to reproducibly generate ions from MCP monomers. While the acidic conditions of MALDI‐TOF MS sample preparation have been shown to aid in disassembly of some phage capsids, many require further treatment to successfully liberate MCP monomers. The findings presented here suggest that β‐mercaptoethanol reduction of the disulfide bonds linking phage MCPs prior to mass spectrometric analysis results in significantly increased MALDI‐TOF MS sensitivity and reproducibility of Yersinia pestis‐specific phage protein profiles. Copyright © 2009 John Wiley & Sons, Ltd.     

Biotransformation and metabolic profile of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In our previous studies, caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐ cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) technique combined with Metabolynx^TMsoftware was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF‐MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant. Copyright © 2015 John Wiley & Sons, Ltd.     

Differential characterization of biogenic amine‐producing bacteria involved in food poisoning using MALDI‐TOF mass fingerprinting

Histamine poisoning is caused by the consumption of fish and other foods that harbor bacteria possessing histidine decarboxylase activity. With the aim of preventing histamine formation, highly specific mass spectral fingerprints were obtained from the 16 major biogenic amine‐producing enteric and marine bacteria by means of MALDI‐TOF MS analysis. All bacterial strains analyzed exhibited specific spectral fingerprints that enabled its unambiguous differentiation. This technique also identified peaks common to certain bacterial groups. Thus, two protein peaks at m/z 4182±1 and 8363±6 were found to be present in all Enterobacteriaceae species analyzed except for Morganella morganii. Peaks at m/z 3635±1 and 7267±2 were specific to both M. morganii and Proteus spp. Biogenic amine‐forming Proteus spp. exhibited three genus‐specific peaks at m/z 3980, 7960±1 and 9584±2. The genus Photobacterium also showed three genus‐specific peaks at m/z 2980±1, 4275±1 and 6578±1. The two histamine‐producing Gram‐positive bacteria Lactobacillus sp. 30A and Staphylococcus xylosus exhibited a few protein peaks in the 2000–7000 m/z range and could be easily distinguished from biogenic amine‐forming Gram‐negative bacteria. Clustering based on MALDI‐TOF MS also exhibited a good correlation with phylogenetic analysis based on the 16S rRNA gene sequence, validating the ability of the MALDI‐TOF technique to establish relationships between microbial strains and species. The approach described in this study leads the way toward the rapid and specific identification of major biogenic amine‐forming bacteria based on molecular protein markers with a goal to the timely prevention of histamine food poisoning.     

Two New Dammarane‐Type Triterpenoid Saponins from Gynostemma pentaphyllum

Two new dammarane‐type triterpenoid saponins were isolated from the EtOH extract of Gynostemma pentaphyllum (Thunb .) Makino . Their structural elucidations were accomplished mainly on the basis of the interpretation of spectroscopic data, such as IR, NMR, and HR‐TOF‐MS. Their liver fibrosis inhibitory activities were evaluated against hepatic stellate cells using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay. Thus, G. pentaphyllum can be used as ingredient for ancillary drugs or functional food.     

Identification of isoquercitrin metabolites produced by human intestinal bacteria using UPLC‐Q‐TOF/MS

In this paper, ultraperformance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS) and the MetaboLynx? software combined with mass defect filtering were applied to identity the metabolites of isoquercitrin using an intestinal mixture of bacteria and 96 isolated strains from human feces. The human incubated samples collected for 72 h in the anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF/MS within 10 min. The parent compound and five metabolites were identified by eight isolated strains, including Bacillus sp. 17, Veillonella sp. 23 and 32 and Bacteroides sp. 40, 41, 56, 75 and 88 in vitro. The results indicate that quercetin, acetylated isoquercitrin, dehydroxylated isoquercitrin, hydroxylated quercetin and hydroxymethylated quercetin are the major metabolites of isoquercitrin. Furthermore, a possible metabolic pathway for the biotransformation of isoquercitrin was established in intestinal flora. This study will be helpful for understanding the metabolic route of isoquercitrin and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and Evaluation of Novel 5‐cyclohexyl‐2‐(4″‐substitutedphenyl)‐3‐(2″‐substitutedphenyl)4H‐2,3,3a,5,6,6a‐hexahydropyrrolo[3,4‐d]isoxazole‐4,6‐dione Derivatives for Their In Vitro Antioxidant and Antibacterial Activities

1,3‐Dipolar cycloaddition reactions of N‐cyclohexyl maleimide ( 1 ) with azomethine N‐oxide ( 2 ) have afforded novel isoxazolidine ( 3 ) in excellent yield. Their structures have been characterized from their IR, ¹H‐NMR, ¹³C‐NMR, ¹H,¹H‐COSY, MS(ESI), and elemental analysis techniques. In vitro antibacterial activity of the synthesized compounds were investigated against a representative panel of pathogenic strains specifically two Gram‐positive bacteria (Staphylococcus aureus and Streptococcus pyogenes ) and two Gram‐negative bacteria (Pseudomonas aeruginosa and Escherichia coli ) using agar‐well diffusion assay. Some of the compounds ( 3a , 3k , 3n , and 3o ) exhibited promising antibacterial activities. All the synthesized compounds have also been screened for their antioxidant activities and were found to be significantly active.     

The effect of temperature on the stability of compounds used as UV‐MALDI‐MS matrix: 2,5‐dihydroxybenzoic acid, 2,4,6‐trihydroxyacetophenone, α‐cyano‐4‐hydroxycinnamic acid, 3,5‐dimethoxy‐4‐hydroxycinnamic acid,nor‐harmane and harmane

The thermal stability of several commonly used crystalline matrix‐assisted ultraviolet laser desorption/ionization mass spectrometry (UV‐MALDI‐MS) matrices, 2,5‐dihydroxybenzoic acid (gentisic acid; GA), 2,4,6‐trihydroxyacetophenone (THA), α‐cyano‐4‐hydroxycinnamic acid (CHC), 3,5‐dimethoxy‐4‐hydroxycinnamic acid (sinapinic acid; SA), 9H‐pirido[3,4‐b]indole (nor‐harmane; nor‐Ho), 1‐methyl‐9H‐pirido[3,4‐b]indole (harmane; Ho), perchlorate of nor‐harmanonium ([nor‐Ho + H]⁺) and perchlorate of harmanonium ([Ho + H]⁺) was studied by heating them at their melting point and characterizing the remaining material by using different MS techniques [electron ionization mass spectrometry (EI‐MS), ultraviolet laserdesorption/ionization‐time‐of‐flight‐mass spectrometry (UV‐LDI‐TOF‐MS) and electrospray ionization‐time‐of‐flight‐mass spectrometry (ESI‐TOF‐MS)] as well as by thin layer chromatography analysis (TLC), electronic spectroscopy (UV‐absorption, fluorescence emission and excitation spectroscopy) and ¹H nuclear magnetic resonance spectroscopy (¹H‐NMR). In general, all compounds, except for CHC and SA, remained unchanged after fusion. CHC showed loss of CO₂, yielding the trans‐/cis‐4‐hydroxyphenylacrilonitrile mixture. This mixture was unambiguously characterized by MS and ¹H‐NMR spectroscopy, and its sublimation capability was demonstrated. These results explain the well‐known cluster formation, fading (vanishing) and further recovering of CHC when used as a matrix in UV‐MALDI‐MS. Commercial SA (SA 98%; trans‐SA/cis‐SA 5 : 1) showed mainly cis‐ to‐trans thermal isomerization and, with very poor yield, loss of CO₂, yielding (3′,5′‐dimethoxy‐4′‐hydroxyphenyl)‐1‐ethene as the decarboxilated product. These thermal conversions would not drastically affect its behavior as a UV‐MALDI matrix as happens in the case of CHC. Complementary studies of the photochemical stability of these matrices in solid state were also conducted. Copyright © 2008 John Wiley & Sons, Ltd.     

Direct identification of trypanosomatids by matrix‐assisted laser desorption ionization–time of flight mass spectrometry (DIT MALDI‐TOF MS)

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix‐assisted laser desorption ionization/time of flight mass spectrometry (MALDI‐TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI‐TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI‐TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI‐TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software‐assisted identification at the strain level. Overall, this study shows the importance of MALDI‐TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry‐based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.     

Block length determination of the block copolymer mPEG‐b‐PS using MALDI‐TOF MS/MS

The molar mass determination of block copolymers, in particular amphiphilic block copolymers, has been challenging with chromatographic techniques. Therefore, methoxy poly(ethylene glycol)‐b‐poly(styrene) (mPEG‐b‐PS) was synthesized by atom transfer radical polymerization (ATRP) and characterized in detail not only by conventional chromatographic techniques, such as size exclusion chromatography (SEC), but also by matrix‐assisted laser/desorption ionization tandem mass spectrometry (MALDI‐TOF MS/MS). As expected, different molar mass values were obtained in the SEC measurements depending on the calibration standards (either PEG or PS). In contrast, MALDI‐TOF MS/MS analysis allowed the molar mass determination of each block, by the scission of the weakest point between the PEG and PS block. Thus, fragments of the individual blocks could be obtained. The PEG block showed a depolymerization reaction, while for the PS block fragments were obtained in the monomeric, dimeric, and trimeric regions as a result of multiple chain scissions. The block length of PEG and PS could be calculated from the fragments recorded in the MALDI‐TOF MS/MS spectrum. Furthermore, the assignment of the substructures of the individual blocks acquired by MALDI‐TOF MS/MS was accomplished with the help of the fragments that were obtained from the corresponding homopolymers. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Bioactivity‐based ultra‐performance liquid chromatography‐coupled quadrupole time‐of‐flight mass spectrometry for NF‐κB inhibitors identification in Chinese Medicinal Preparation Bufei Granule

Traditional Chinese medicine (TCM) preparations have become effective treatments for many diseases. However, their active ingredients are still uncertain and difficult to identify. In this study, we propose a strategy that integrates ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) and bioactive (NF‐κB inhibitor) luciferase reporter assay systems for the rapid determination of various anti‐inflammatory compounds of TCM preparations. In this way, Bufei Granule (BFG), a TCM preparation used for the clinical therapy of asthma, was analyzed by the two ways of component identification and activity detection. Potential anti‐inflammatory constituents were screened by NF‐κB activity assay systems and simultaneously identified according to the mass spectrometry data. Three structural types of NF‐κB inhibitors (caffeic acid derivatives, flavonoids and Pentacyclic triterpenes) were characterized. Further cytokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional chromatographic separation and inhibitory activity detection, integrating UPLC/Q‐TOF‐MS identification and virtual validation was more convenient and more reliable. This strategy clearly demonstrates that MS data‐based fingerprinting is a meaningful tool not only in identifying constituents in complex matrix but also in directly screening for powerful trace ingredients in TCM preparations. Copyright © 2016 John Wiley & Sons, Ltd.     

Sodiation as a tool for enhancing the diagnostic value of MALDI‐TOF/TOF‐MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono‐ and diesters. For rapid fingerprinting of these esters, matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF/TOF‐MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono‐ and diester palmitate standards in MALDI‐TOF/TOF‐MS showed that sodium adduct parent masses [M + Na]⁺ gave much simpler MS² spectra than radical / protonated [M]^+● / [M + H]⁺ parents. [M + Na]⁺ fragments yielded diagnostic polyene‐specific eliminations and fatty acid neutral losses, whereas [M]^+● / [M + H]⁺ fragmentation resulted in a multitude of non‐diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na]⁺ ionization by addition of sodium acetate, and best signal‐to‐noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI‐TOF/TOF‐MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono‐ and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all‐trans esterified esters found in LC were identified with MALDI‐TOF/TOF‐MS, with the exception of two minor monoesters. Copyright © 2013 John Wiley & Sons, Ltd.     

Affinity capture using chimeric membrane proteins bound to magnetic beads for rapid ligand screening by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K⁺ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore‐forming region of human K⁺ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal‐bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity‐captured scorpion toxins were eluted prior to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS), and to nano‐electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity‐capture screening assay led to the isolation and characterization of potent and specific ligands of the human K⁺ channel, Kv1.3. The affinity‐capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re‐used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure. Copyright © 2009 John Wiley & Sons, Ltd.