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.     

High mass accuracy in-source collision-induced dissociation tandem mass spectrometry and multi-step mass spectrometry as complementary tools for fragmentation studies of quaternary ammonium herbicides

Fragmentation studies using both an ion-trap mass analyzer and a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer were performed in order to establish the fragmentation pathways of organic molecules. A general strategy combining MSn data (n = 1-4) in an ion-trap analyzer with tandem mass spectrometry and in-source collision-induced dissociation tandem mass spectrometry (CID MS/MS) in a Q-TOF instrument was applied. The MSn data were used to propose a tentative fragmentation pathway following genealogical relationships. When several assignments were possible, MS/MS and in-source CID MS/MS (Q-TOF) allowed the elemental compositions of the fragments to be confirmed. Quaternary ammonium herbicides (quats) were used as test compounds and their fragmentation pathways were established. The elemental composition of the fragments was confirmed using the TOF analyzer with relative errors <0.0023 Da. Some fragments previously reported in the literature were reassigned taking advantage of the high mass resolution and accuracy of the Q-TOF instrument, which made it possible to solve losses where nitrogen was involved.     

Critical assessment of ionization patterns and applications of ambient desorption/ionization mass spectrometry using FAPA–MS

Ambient desorption/ionization mass spectrometry (MS) has gained growing interest during the last decade due to its high analytical performance and yet simplicity. Here, one of the recently developed ambient desorption/ionization MS sources, the flowing atmospheric‐pressure afterglow (FAPA) source, was investigated in detail regarding background ions and typical ionization patterns in the positive as well as the negative ion mode for a variety of compound classes, comprising alkanes, alcohols, aldehydes, ketones, carboxylic acids, organic peroxides and alkaloids. A broad range of signals for adducts and losses was found, besides the usually emphasized detection of quasimolecular ions, i.e. [M + H]⁺ and [M ? H]^? in the positive and the negative mode, respectively. It was found that FAPA–MS is best suited for polar analytes containing nitrogen and/or oxygen functionalities, e.g. carboxylic acids, with low molecular weights and relatively high vapor pressures. In addition, the source was used in proof‐of‐principle studies, illustrating the capabilities and limitations of the technique: Firstly, traces of cocaine were detected and unambiguously identified on euro banknotes using FAPA ionization in combination with tandem MS, suggesting a correlation between cocaine abundance and age of the banknote. Secondly, FAPA–MS was used for the identification of acidic marker compounds in organic aerosol samples, indicating yet‐undiscovered matrix and sample surface effects of ionization pathways in the afterglow region. Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluation of the efficiency of the concurrent use of IR and mass spectrometry databases for structure elucidation

The applicability of structural analogy to structure elucidation of organic compounds by searching two molecular spectroscopy databases (DBs) is examined. Using structural analogy is based on the representation of DB structures as sets of structural fragments. Of primary concern are the structural fragments that are represented in the search results of both infrared (IR) and mass spectroscopy (MS) DBs. The statistically justified estimates of the efficiency of the combined search depending on the spectral similarity are given.     

ESI,APCI, and MALDI tandem mass spectrometry of poly(methyl acrylate)s: A comparison study for the structural characterization of polymers synthesized via CRP techniques and the software application to analyze MS/MS data

Research in polymer science and engineering is moving from classical methodologies to advanced analytical strategies in which mass spectrometry (MS)‐based techniques play a crucial role. The molecular complexity of polymers requires new characterization tools and approaches to elucidate the detailed structural information. In this contribution, a comparison study of poly(methyl acrylate)s (PMA) using different tandem mass spectrometry techniques (ESI, APCI, and MALDI MS/MS) is reported to provide insights into the macromolecular structure with the aid of a special MS/MS data interpretation software. Collision‐induced dissociation (CID) was utilized to examine the fragmentation pathways of PMAs synthesized via various controlled radical polymerization techniques. All three mass spectrometry techniques are used to analyze structural details of PMAs and the labile end‐groups are determined based on the fragmentation behavior in CID. Fragmentation products were identified which are characteristics for the cleavage between the polymer chain and the end‐group. The application of a tailor‐made software is shown to analyze complex MS/MS data, and it is proven that this kind of software will be helpful for polymer scientists to identify fragmentation products obtained by tandem mass spectrometry similar to the fields of proteomics, metabolomics, genomics, and glycomics. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Low‐energy collision‐induced dissociation (low‐energy CID), collision‐induced dissociation (CID), and higher energy collision dissociation (HCD) mass spectrometry for structural elucidation of saccharides and clarification of their dissolution mechanism in DMAc/LiCl

The dissolution mechanism of oligosaccharides in N,N‐dimethylacetamide/lithium chloride (DMAc/LiCl), a solvent used for cellulose dissolution, and the capabilities of low‐energy collision‐induced dissociation (low‐energy CID), collision‐induced dissociation (CID), and higher energy collision dissociation (HCD) for structural analysis of carbohydrates were investigated. Comparing the spectra obtained using 3 techniques shows that, generally, when working with monolithiated sugars, CID spectra provide more structurally informative fragments, and glycosidic bond cleavage is the main pathway. However, when working with dilithiated sugars, HCD spectra can be more informative providing predominately cross‐ring cleavage fragments. This is because HCD is a nonresonant activation technique, and it allows a higher amount of energy to be deposited in a short time, giving access to more endothermic decomposition pathways as well as consecutive fragmentations. The difference in preferred dissociation pathways of monolithiated and dilithiated sugars indicates that the presence of the second lithium strongly influences the relative rate constants for cross‐ring cleavages vs glycosidic bond cleavages, and disfavors the latter. Regarding the dissolution mechanism of sugars in DMAc/LiCl, CID and HCD experiments on dilithiated and trilithiated sugars reveal that intensities of product ions containing 2 Li⁺ or 3 Li⁺, respectively, are higher than those bearing only 1 Li⁺. In addition, comparing the fragmentation spectra (both HCD and CID) of LiCl‐adducted lithiated sugar and NaCl‐adducted sodiated sugar shows that while, in the latter case, loss of NaCl is dominant, in the former case, loss of HCl occurs preferentially. The compiled evidence implies that there is a strong and direct interaction between lithium and the saccharide during the dissolution process in the DMAc/LiCl solvent system.     

Characterization of complex,heterogeneous lipid A samples using HPLC‐MS/MS technique III. Positive‐ion mode tandem mass spectrometry to reveal phosphorylation and acylation patterns of lipid A

In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low‐energy collision‐induced dissociation conditions of an electrospray quadrupole time‐of‐flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed‐phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive‐ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter‐ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4′ phosphate group) were simultaneously present in the sample. Continuous series of high‐abundance B‐type and low‐abundance Y‐type inter‐ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative‐ion mode, they were very similar in the positive‐ion mode. The complementary use of positive‐ion and negative‐ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1‐monophosphorylated lipid A species.     

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and hydrogen exchange combined with enzymatic digestion for the structural characterization of antimalaric Spf66 peptide

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used in hydrogen exchange studies, exchanging deuteron (H/D) or proton (D/H), to determine the structure and conformational changes of antimalarial Spf66 synthetic peptide in its monomeric and dimeric forms. The accuracy of both analytical methods was assessed along with their suitability to study structural aspects. The results via these two approaches were in agreement, indicating that the dimer presents segments of secondary structure. In this last case, the combination of both methods with enzymatic digestion with pepsin was used in their identification. Although 100% coverage of Spf66 dimer was not observed, the higher levels of deuteration were observed for fragments located in the chain terminal where the structure may be more flexible, while the fragments near the disulfide bonds, which is, in theory, the more rigid region of the molecule, were not detected. This strategy is significantly time saving and allows rapid screening and help to characterize a protein, especially, when no prior structural information is available. However, a single spectrum is not certainly sufficient to obtain structural data; it is just an experimental limitation.Also, changes in peptide structure after storage at different temperatures and time were observed, which lead to a loss in the secondary structure as determined by circular dicroism measurements and an increase in aggregation products, since the trimer and tetramer species were detected by mass spectrometry.     

Multiplex liquid chromatography-tandem mass spectrometry for the detection of wheat,oat, barley and rye prolamins towards the assessment of gluten-free product safety

Celiac patients should feel confident in the safety of foods labelled or expected to be gluten-free. In this context, a targeted proteomic approach based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was proposed to assess the presence of celiotoxic cereals, namely wheat, oats, barley and rye, in raw and processed food products. To this aim, unique marker peptides were properly selected in order to distinguish between the different cereal types. A revised cocktail solution based on reducing and denaturing agents was exploited for prolamin extraction from raw and processed food; in addition, defatting with hexane was carried out for sample clean-up, allowing to largely reduce problems related to matrix effect. Method validation on fortified rice flour showed good analytical performance in terms of sensitivity (limits of detection in the 2–18 mg kg⁻¹ range). However, poor trueness was calculated for self-made incurred bread (between 3 and 30% depending on the peptide), probably due to baking processes, which reduce gluten extractability. Thus, it is evident that in the case of processed foods further insights into sample treatment efficiency and reference materials for protein calibration are required to obtain accurate gluten determination. Finally, the developed method was applied for the analysis of market food products, offering the possibility to discriminate among cereals, with good agreement with labelled ingredients for gluten-containing foodstuffs.     

Multi-analyte high performance liquid chromatography coupled to high resolution tandem mass spectrometry method for control of pesticide residues,mycotoxins, and pyrrolizidine alkaloids

A new reliable and highly sensitive method based on high performance liquid chromatographic (HPLC) separation and high resolution tandem mass spectrometric detection (HRMS/MS) has been developed and validated for determination of 323 pesticide residues, 55 mycotoxins, and 11 plant toxins represented by pyrrolizidine alkaloids. The method was validated for three matrices, leek, wheat, and tea differing in nature/amount of co-extracts that may cause various matrix effects. For target analytes isolation, optimized QuEChERS-based (quick, easy, cheap, effective, rugged, and safe) extraction procedure was employed. Spectral HRMS/MS library has been established providing an entire spectrum of fragment ions for each analyte, which allows unbiased identification and confirmation of target compounds. The limits of quantification (LOQs) of target analytes were below 10 μg kg⁻¹ for 82%, 81%, and 61% for matrices leek, wheat, and tea, respectively. Recoveries were in the acceptable range (70–120%) according to SANCO/12571/2013 for most of target analytes, except for highly polar ‘masked’ mycotoxin deoxynivalenol-3-glucoside with recoveries 35%, 47%, and 42% for matrices leek, wheat, and tea, respectively. The linearities of calibration curves expressed as coefficients of determination were in the range of 0.9661–1.000, and repeatabilities expressed as relative standard deviations (RSDs) at LOQs lied in the range of 0.25–13.51%. The trueness of the method was verified using several certified reference materials (CRMs) and proficiency test samples.