Gangliosides from human granulocytes: a nano-ESI QTOF mass spectrometry fucosylation study of low abundance species in complex mixtures

Nano-ESI QTOF MS was used for sensitive mapping and sequencing of single molecular species in complex ganglioside mixtures obtained from human granulocytes, where the fucosylated carbohydrate chains of granulocyte gangliosides carry sLex and VIM-2 epitopes postulated to interact with E-selectin of the blood vessel wall in the early phase of the inflammation process. Functionally relevant components are expressed only at a low level, but using the negative ion detection it is possible to trace and identify such species, which were not detectable even by TLC. Advantage of the low-energy CID fragmentation for low abundance components of the complex ganglioside mixtures in the negative ion mode is to produce clear-cut series of fragment ions for sequencing. Fucosylation analysis carried out for each molecular species by MS/MS permits the clear distinction between sLex and VIM-2 epitope. VIM-2 epitope was expressed in all species with a longer sugar core, while in the short oligosaccharide chain with a sLex only, using biological material at a mid-femtomol level detection.     

Fully-automated chip-based nanoelectrospray tandem mass spectrometry of gangliosides from human cerebellum

The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is described.     

Glycoscreening by on-line sheathless capillary electrophoresis/electrospray ionization-quadrupole time of flight-tandem mass spectrometry

An analytical approach based on sheathless on-line coupling of capillary electrophoresis (CE) and electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been developed for providing new insight into the characterization of carbohydrate mixtures. The home-built sheathless CE/     

Off-line capillary electrophoresis/fully automated nanoelectrospray chip quadrupole time-of-flight mass spectrometry and tandem mass spectrometry for glycoconjugate analysis

Implementation and optimization of an off-line capillary electrophoresis (CE)/(−)nanoESIchip-quadrupole time-of-flight (QTOF) mass spectrometric (MS) and tandem MS system for compositional mapping and structural investigation of components in complex carbohydrate mixtures is described. The approach was developed for glycoscreening and applied to O-glycosylated peptides from urine of a patient suffering from α-N-acetylhexosaminidase deficiency, known as Schindler's disease. The fundamental issue of sensitivity, previously representing a serious drawback of the off-line CE/MS analysis, could be positively addressed by the off-line conjunction of CE with automated chip-based ESI-QTOF-MS to provide flexibility for CE/chip MS coupling and enhance structural elucidation of single components in heterogeneous mixtures. Copyright © 2004 John Wiley & Sons, Ltd.     

Quantitative analysis of hindered amine light stabilizers by CZE with UV detection and quadrupole TOF mass spectrometric detection

The current work describes the development of a CZE method with quadrupole QTOF‐MS detection and UV detection for the quantitation of Cyasorb 3529, a common hindered amine light stabilizer (HALS), in polymer materials. Analysis of real polymer samples revealed that the oligomer composition of Cyasorb 3529 changes during processing, a fact hampering the development of a straightforward method for quantitation based on calibration with a Cyasorb 3529 standard. To overcome this obstacle in‐depth investigations of the oligomer composition of this HALS using QTOF‐MS and QTOF‐MS/MS had to be performed whereby 22 new oligomer structures, in addition to the ten structures already described, were identified. Finally, a CZE method for quantitative analysis of this HALS was developed starting with a comprehensive characterization of a Cyasorb 3529 standard using CZE‐QTOF‐MS, subsequently allowing the correct assignment of most Cyasorb 3529 oligomers in an electropherogram with UV detection. Employing the latter detection technique and hexamethyl‐melamine as internal standard, peak areas obtained for the melamine could be correlated with those from the triazine ring, the UV‐absorbing unit present in the HALS. This approach finally allowed proper quantitation of the single oligomers of Cyasorb 3529, an imperative for the quantitative assessment of this HALS in real polymer samples.     

Characterization of O-glycosylation sites in MUC2 glycopeptides by nanoelectrospray QTOF mass spectrometry

Sequencing of eight O-glycosylated peptides by nanoESI-QTOF-MS/MS was carried out to provide a sensitive general characterization method for determination of glycosylation site(s) and of the type of the attached carbohydrate moiety in a single experiment. The glycopeptide structures were chosen to demonstrate the feasibility of this sensitive and accurate approach, where isobaric peptide structures either (i) with the same number of attachment sites in different position in the peptide backbone, and (ii) with the same number of sugar moieties distributed on different attachment sites in the peptide backbone, can be clearly distinguished. Beside the B-type carbohydrate sequence ions of high abundance, it is possible to register diagnostic b- and y-type glycosylated peptide ions of lower abundance due to high dynamic range of the QTOF analyser. The applicability of this approach for detailed analysis of highly clustered O-glycan structures as found in biological mucin samples is discussed.     

液相色谱-质谱联用方法用于严重急性呼吸系统综合症冠状病毒结构蛋白质的研究

通过高效液相反相色谱、毛细管反相色谱-串联质谱联用方法对SARS病毒攻击细胞的研究,鉴定出了N、S和M3种SARS结构蛋白质,并准确测定了N蛋白质的分子量。由分子量结果和生物信息学推断的N蛋白质的理论分子量比较,可以确定N蛋白质不存在常见的磷酸化修饰和糖基化修饰或含量很低。进一步的研究表明：N蛋白质可能存在降解现象,其降解机理尚需探索。另外,通过对样品直接酶切后进行两维毛细管分离和串联质谱鉴定与对样品先进行分离,然后再进行毛细管反相分离-串联质谱鉴定方法比较,表明两种方法在蛋白质组学研究中各具优缺点,可根据不同目的选择使用。     

A strategy for direct identification of protein S-nitrosylation sites by quadrupole time-of-flight mass spectrometry

S-nitrosylation of proteins serves an important role in regulating diverse cellular processes including signal transduction, DNA repair, and neurotransmission. Identification of S-nitrosylation sites is crucial for understanding the significance of this post-translational modification (PTM) in modulating the function of a protein. However, it is challenging to identify S-nitrosylation sites directly by mass spectrometric (MS) methods due to the labile nature of the S-NO bond. Here we describe a strategy for direct identification of protein S-nitrosylation sites in an electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometer without prior chemical derivatization of S-nitrosylated peptides. Both sample buffer composition and MS hardware parameters were carefully adjusted to ensure that S-nitrosylated peptide ions could be analyzed by the QTOF MS with optimal signal/noise ratios. It was crucial that the proteins were preserved in a sample solution containing 1 mM EDTA and 0.1 mM neocuproine at neutral pH. Proteins dissolved in this solution are amenable to in-solution tryptic digestion, which is important for the analysis of biological samples. S-nitrosylated peptides were effectively analyzed by LC/MS/MS on QTOF MS, with an optimized cone voltage of 20 V and collision energy of 4 V. We have successfully applied this method to thioredoxin, a key antioxidant protein, and identified within it an S-nitrosylation site at Cys73.     

Joint MS‐based platforms for comprehensive comparison of rat plasma and serum metabolic profiling

Metabolomics is a rapidly growing field in the comprehensive understanding of cellular and organism‐specific responses associated with perturbations induced by medicines, chemicals and environment. Blood matrices are frequently used in clinical and biological studies. In this study, we compared metabolic profiling between rat plasma and serum using complementary platforms of gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography–quadruple time‐of‐flight–mass spectrometry (LC‐QTOF‐MS). The sample types that were tested included plasma prepared with K₂EDTA and serum collected using venous blood collection protocols. The results of peak area variation for each detected metabolite/feature in the quality control samples showed a good reproducibility in LC‐QTOF‐MS and better reproducibility in GC‐MS. In GC‐MS analysis: (a) 25.8% of the defined metabolites differed serum from plasma profiling (t‐test, p < 0.05); and (b) serum possessed higher sensitivity than plasma for its generally higher peak intensity in the metabolic profiling. In LC‐QTOF‐MS analysis, 13 (in positive ion mode) and seven (in negative ion mode) important metabolites were identified as mainly contributing to the separation between serum and plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Sensitive LC MS quantitative analysis of carbohydrates by Cs<Superscript>+</Superscript> attachment

The development of a sensitive assay for the quantitative analysis of carbohydrates from human plasma using LC/MS/MS is described in this paper. After sample preparation, carbohydrates were cationized by Cs(+) after their separation by normal phase liquid chromatography on an amino based column. Cesium is capable of forming a quasi-molecular ion [M + Cs](+) with neutral carbohydrate molecules in the positive ion mode of electrospray ionization mass spectrometry. The mass spectrometer was operated in multiple reaction monitoring mode, and transitions [M + 133] --> 133 were monitored (M, carbohydrate molecular weight). The new method is robust, highly sensitive, rapid, and does not require postcolumn addition or derivatization. It is useful in clinical research for measurement of carbohydrate molecules by isotope dilution assay.     

Electrospray ionization collision‐induced dissociation mass spectrometry: a tool to characterize synthetic polyaminocarboxylate ferric chelates used as fertilizers

Fertilizers based on synthetic polyaminocarboxylate ferric chelates have been known since the 1950s to be successful in supplying Fe to plants. In commercial Fe(III)‐chelate fertilizers, a significant part of the water‐soluble Fe‐fraction consists of still uncharacterized Fe byproducts, whose agronomical value is unknown. Although collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS) is a valuable tool for the identification of such compounds, no fragmentation data have been reported for most Fe(III)‐chelate fertilizers. The aim of this study was to characterize the CID‐MS² fragmentation patterns of the major synthetic Fe(III)‐chelates used as Fe‐fertilizers, and subsequently use this technique for the characterization of commercial fertilizers. Quadrupole‐time‐of‐flight (QTOF) and spherical ion trap mass analyzers equipped with an electrospray ionization (ESI) source were used. ESI‐CID‐MS² spectra obtained were richer when using the QTOF device. Specific differences were found among Fe(III)‐chelate fragmentation patterns, even in the case of positional isomers. The analysis of a commercial Fe(III)‐chelate fertilizer by high‐performance liquid chromatography (HPLC) coupled to ESI‐MS(QTOF) revealed two previously unknown, Fe‐containing compounds, that were successfully identified by a comprehensive comparison of the ESI‐CID‐MS²(QTOF) spectra with those of pure chelates. This shows that HPLC/ESI‐CID‐MS²(QTOF), along with the Fe(III)‐chelate fragmentation patterns, could be a highly valuable tool to directly characterize the water‐soluble Fe fraction in Fe(III)‐chelate fertilizers. This could be of great importance in issues related to crop Fe‐fertilization, both from an agricultural and an environmental point of view. Copyright © 2009 John Wiley & Sons, Ltd.     

Building an empirical mass spectra library for screening of organic pollutants by ultra-high-pressure liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

Hybrid quadrupole time-of-flight mass spectrometry (QTOF MS) has gained wide acceptance in many fields of chemistry, for example, proteomics, metabolomics and small molecule analysis. This has been due to the numerous technological advances made to this mass analyser in recent years. In the environmental field, the instrument has proven to be one of the most powerful approaches for the screening of organic pollutants in different matrices due to its high sensitivity in full acquisition mode and mass accuracy measurements. In the work presented here, the optimum experimental conditions for the creation of an empirical TOF MS spectra library have been evaluated. For this model we have used a QTOF Premier mass spectrometer and investigated its functionalities to obtain the best MS data, mainly in terms of mass accuracy, dynamic range and sensitivity. Different parameters that can affect mass accuracy, such as lock mass, ion abundance, spectral resolution, instrument calibration or matrix effect, have also been carefully evaluated using test compounds (mainly pesticides and antibiotics). The role of ultra-high-pressure liquid chromatography (UHPLC), especially when dealing with complex matrices, has also been tested. In addition to the mass accuracy measurements, this analyser allows the simultaneous acquisition of low and high collision energy spectra. This acquisition mode greatly enhances the reliable identification of detected compounds due to the useful (de)protonated molecule and fragment ion accurate mass information obtained when working in this mode. An in-house empirical spectral library was built for approximately 230 organic pollutants making use of QTOF MS in MS(E) mode. All the information reported in this paper is made available to the readers to facilitate screening and identification of relevant organic pollutants by QTOF MS.     

Rapid screening and characterization of metabolites from a marine-derived actinomycete by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry

A rapid and reliable method has been optimized and established for the analysis of the metabolites from a marine actinomycete by high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (HPLC/QTOF MS/MS). From MS/MS spectra, the product ions of [M + H]⁺ were recorded to provide abundant structural information of the mother nucleus and peptide moieties. Using the QTOF MS/MS and in‐source collision‐induced dissociation (in‐source CID) techniques, three main metabolites including actinomycin D, actinomycin V and actinomycin I were determined and characterized by elemental compositions of precursor and product ions (<7 ppm). Additionally, this method provided information about the compositions of the peptide residues and the sequences of the amino acid from a series of fragment ions. It proved useful for the identi?cation of the metabolites in marine samples which have similar structures especially when there were no reference compounds available. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification,confirmation and screening capability of UHPLC coupled to triple quadrupole and hybrid quadrupole time‐of‐flight mass spectrometry in pesticide residue analysis

The potential of three mass spectrometry (MS) analyzers (triple quadrupole, QqQ; time of flight, TOF; and quadrupole time of flight, QTOF) has been investigated and compared for quantification, confirmation and screening purposes in pesticide residue analysis of fruit and vegetable samples. For this purpose, analytical methodology for multiresidue determination of 11 pesticides, taken as a model, has been developed and validated in nine food matrices for the three mass analyzers coupled to ultra high pressure liquid chromatography. In all cases, limits of quantification around 0.01 mg/kg were reached, fulfilling the most restrictive case of baby‐food analysis. Regarding absolute sensitivity, the lower limits of detection were obtained, as expected, for QqQ (100 fg), whereas slightly higher limits (300 fg) were obtained for both TOF and QTOF. Confirmative capacity of each analyzer was studied for each analyte based on the identification points (IPs) criterion, useful for a comprehensive comparison. QTOF mass analyzer showed the highest confirmatory capacity, although QqQ normally led to sufficient number of IPs, even at lower concentration levels. The potential of TOF MS was also investigated for screening purposes. To this aim, around 50 commercial fruits and vegetables samples were analyzed, searching for more than 400 pesticides. TOF MS proved to be an attractive analytical tool for rapid detection and reliable identification of a large number of pesticides thanks to the full spectrum acquisition at accurate mass with satisfactory sensitivity. This process is readily boosted when combined with specialized software packages, together with theoretical exact mass databases. Several pesticides (e.g. carbendazim in citrus and indoxacarb in grape) were detected in the samples. Further unequivocal confirmation of the identity was performed using reference standards and/or QTOF MS/MS experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid simultaneous screening and identification of multiple pesticide residues in vegetables

A method for the rapid simultaneous screening and identification of multiple pesticide residues in vegetables was established using a novel database and gas chromatography in combination with hybrid quadrupole time-of-flight mass spectrometry (GC–QTOF MS). A total of 187 pesticides with different chemical species were measured by GC–QTOF MS to create the database, which collected the retention time and exact masses of ions from the first-stage mass spectrum (MS¹ spectrum) and second-stage mass spectrum (MS² spectrum) for each pesticide. The workflow of the created database consisted of “MS¹ screening” for possible pesticides by chemical formula match and “MS² identification” for structural confirmation of product ion by accurate mass measurement. To evaluate the applicability of the database, a spinach matrix was prepared by solid phase extraction, spiked with a mixture of 50 pesticides at seven concentrations between 0.1 and 10 ppb, and analyzed by GC–QTOF MS. It was found that all of the 50 pesticides with concentrations as low as 5 ppb were detected in the “MS¹ screening” step and accurate masses were identified with errors less than 2.5 mDa in the “MS² identification” step, indicating high sensitivity, accuracy, selectivity and specificity. Finally, to validate the applicability, the new method was applied to four fresh celery, rape, scallion and spinach vegetables from a local market. As a result, a total of 13 pesticides were found, with 11 in celery, 9 in rape, 3 in scallion and 2 in spinach. In conclusion, GC–QTOF MS combined with an exact mass database is one of the most efficient tools for the analysis of pesticide residues in vegetables.     

Sequencing of tri- and tetraantennary N-glycans containing sialic acid by negative mode ESI QTOF tandem MS

Application of the negative mode electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI QTOF) tandem MS for determination of substitution patterns by sialic acid and/or fucose and extention by additional LacNAc disaccharide units in single branches of multianternary N-glycans from biological samples is described. Fragmentation patterns which can be obtained by low energy collision-induced dissociation (CID) using the QTOF instrument include cleavage ions, diagnostic for determination of antennarity and for specific structural features of single antennae. Systematic fragmentation studies in the negative ion mode were focussed toward formation of the D diagnostic ion relevant for assignment of 3- and 6-antennae in complex N-glycans carrying three and four antennae in combination with epitope-relevant B- and C-type ions. For validation of this approach ESI QTOF fragmentation of the permethylated analogues was carried out in the positive ion mode. Using this strategy, products of in vitro glycosylation reactions were investigated in order to clarify some general aspects of N-glycan acceptor specificity during biosynthesis. Alpha1-3fucosylation using GDP-fucose along with a soluble form of the recombinant human alpha1-3fucosyltransferase VI was carried out on tri- and tetraantennary precursors to test structural requirements for formation of Le(x) versus sLe(x) motifs.     

New insights on proteomics of transgenic soybean seeds: evaluation of differential expressions of enzymes and proteins

This work reports the evaluation of differentially expressed enzymes and proteins from transgenic and nontransgenic soybean seeds. Analysis of malondialdehyde, ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), and catalase (EC 1.11.1.6) revealed higher levels (29.8, 30.6, 71.4, and 35.3%, respectively) in transgenic seeds than in nontransgenic seeds. Separation of soybean seed proteins was done by two-dimensional polyacrylamide gel electrophoresis, and 192 proteins were identified by matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and electrospray ionization (ESI) QTOF MS. Additionally, the enzyme CP4 EPSPS, involved in the genetic modification, was identified by enzymatic digestions using either trypsin or chymotrypsin and ESI-QTOF MS/MS for identification. From the proteins identified, actin fragment, cytosolic glutamine synthetase, glycinin subunit G1, and glycine-rich RNA-binding protein were shown to be differentially expressed after analysis using the two-dimensional difference gel electrophoresis technique, and applying a regulator factor of 1.5 or greater.     

Potential of liquid chromatography/time-of-flight mass spectrometry for the determination of pesticides and transformation products in water

Until now, time-of-flight (TOF) mass analysers have only been very rarely used in pesticide residue analysis (PRA) of water samples. However, the inherent characteristics of TOF MS make these analysers well-suited to this field, mainly for qualitative purposes. Thus, the high sensitivity obtained from full-scan acquisition in comparison to other MS analysers and the high resolution of TOF MS suggest its suitability for screening purposes; it also increases the multiresidue capabilities of methods based on it and decreases the chance of recording false positives. Although these characteristics can also be helpful for quantification, confirmation and elucidation, some limitations on the use of TOF for these purposes have been observed. These limitations are more noticeable when dealing with samples containing very low analyte concentrations, which is the normal situation for PRA in water. The use of hybrid quadrupole–time-of-flight instruments (QTOF) minimises the limitations of TOF, facilitating the simultaneous detection and unequivocal confirmation of pesticides found in the sample. Additionally, the acquisition of accurate product ion full-scan mass spectra can help to elucidate the structures of unknown compounds. In this paper, the potential of TOF and QTOF hyphenated to liquid chromatography for PRA in water is explored, emphasizing both the advantages and limitations of this approach for screening, quantification, confirmation and elucidation purposes. Emphasis is placed on the determination of polar pesticides and transformation products—the analytes that fit well with LC–API–(Q)TOF MS technology.     

Combining size-exclusion chromatography and fully automated chip-based nanoelectrospray quadrupole time-of-flight tandem mass spectrometry for structural analysis of chondroitin/dermatan sulfate in human decorin

Chondroitin/dermatan sulfate (CS/DS) chain of decorin (DCN) from human skin fibroblasts (HSk) was released by reductive β-elimination reaction and digested with chondroitin AC I lyase. Enzymatic hydrolysis mixture of CS/DS chains was separated by size-exclusion chromatography (SEC). Collected octasaccharide fraction was subjected to fully automated chip-based nanoelectrospray (nanoESI) quadrupole time-of-flight (QTOF) MS and tandem MS (MS/MS). MS of human skin fibroblasts DCN CS/DS displayed a high complexity due to the large variety of glycoforms, which under chip-nanoESI MS readily ionized to form multiply charged ions. Except for the regularly tetrasulfated octasaccharide, the investigated fraction contained four additional octasaccharides of atypical sulfation status. Two new oversulfated glycoforms and two undersulfated species were identified. Remarkably, the series of decasaccharides discovered in the same SEC pool was found to encompass a trisulfated and a novel hexasulfated [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?] species. MS/MS by collision-induced dissociation (CID) on the [M-4H]? ion corresponding to the previously not reported [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?](5S) corroborated for a novel motif in which three N-acetylgalactosamine (GalNAc) moieties are monosulfated, 4,5-Δ-GlcA and the first IdoA from the non-reducing end bear one sulfate group each, while the second N-acetylgalactosamine from the reducing end is unsulfated.     

Pressurized CEC coupled with QTOF‐MS for urinary metabolomics

Pressurized CEC (pCEC) coupled with ESI‐QTOF‐MS using a sheathless interface was applied for metabolomics to develop an alternative analytical method for metabolic profiling of complex biofluid samples such as urine. The hyphenated system was investigated with mixed standards and pooled urine samples to evaluate its precision, repeatability, linearity, sensitivity, and selectivity. The applied voltage, mobile phase, and gradient elution were optimized and applied for the analysis of urinary metabolites. Multivariate data analysis was subsequently performed and used to distinguish lung cancer patients from healthy controls successfully. High separation efficiency has been achieved in pCEC due to the EOF. For metabolite identification, the pCEC‐MS separation mechnism was helpful for discriminating the fragment ions of glutamine conjugates from co‐eluted metabolites. Three glutamine conjugates, including phenylacetylglutamine, acylglutamine C8:1, and acylglutamine C6:1 were identified among 16 differential urinary metabolites of lung cancer. Receiver‐operating‐characteristic analysis of acylglutamine C8:1 resulted in an area‐under‐curve value of 0.882. Overall, this work suggests that this pCEC‐ESI‐QTOF‐MS method may provide a novel and useful platform for metabolomic studies due to its superior separation and identification.