Comparison of CID,ETD and metastable atom‐activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision‐induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom‐activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b‐/y‐type ions with abundant neutral losses of the phosphorylation modification. ETD produced c‐/z‐type ions in highest abundance but also showed numerous y‐type ions at a frequency about 50% that of the z‐type ions. The major peaks observed in the ETD spectra correspond to the charge‐reduced product ions and small neutral losses from the charge‐reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15–20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

New hydrophobic L‐amino acid salts: maleates of L‐leucine,L‐isoleucine and L‐norvaline

Crystals of maleates of three amino acids with hydrophobic side chains [L‐leucenium hydrogen maleate, C₆H₁₄NO₂⁺·C₄H₃O₄⁻, (I), L‐isoleucenium hydrogen maleate hemihydrate, C₆H₁₄NO₂⁺·C₄H₃O₄⁻·0.5H₂O, (II), and L‐norvalinium hydrogen maleate–L‐norvaline (1/1), C₅H₁₁NO₂⁺·C₄H₃O₄⁻·C₅H₁₂NO₂, (III)], were obtained. The new structures contain C₂²(12) chains, or variants thereof, that are a common feature in the crystal structures of amino acid maleates. The L‐leucenium salt is remarkable due to a large number of symmetrically non‐equivalent units (Z′ = 3). The L‐isoleucenium salt is a hydrate despite the fact that L‐isoleucine is a nonpolar hydrophobic amino acid (previously known amino acid maleates formed hydrates only with lysine and histidine, which are polar and hydrophilic). The L‐norvalinium salt provides the first example where the dimeric cation L‐Nva...L‐NvaH⁺ was observed. All three compounds have layered noncentrosymmetric structures. Preliminary tests have shown the presence of the second harmonic generation (SGH) effect for all three compounds.     

Positive chemical ionization triple‐quadrupole mass spectrometry and ab initio computational studies of the multi‐pathway fragmentation of phthalates

We report the first positive chemical ionization (PCI) fragmentation mechanisms of phthalates using triple‐quadrupole mass spectrometry and ab initio computational studies using density functional theories (DFT). Methane PCI spectra showed abundant [M + H]⁺, together with [M + C₂H₅]⁺ and [M + C₃H₅]⁺. Fragmentation of [M + H]⁺, [M + C₂H₅]⁺ and [M + C₃H₅]⁺ involved characteristic ions at m/z 149, 177 and 189, assigned as protonated phthalic anhydride and an adduct of phthalic anhydride with C₂H₅⁺ and C₃H₅⁺, respectively. Fragmentation of these ions provided more structural information from the PCI spectra. A multi‐pathway fragmentation was proposed for these ions leading to the protonated phthalic anhydride. DFT methods were used to calculate relative free energies and to determine structures of intermediate ions for these pathways. The first step of the fragmentation of [M + C₂H₅]⁺ and [M + C₃H₅]⁺ is the elimination of [R? H] from an ester group. The second ester group undergoes either a McLafferty rearrangement route or a neutral loss elimination of ROH. DFT calculations (B3LYP, B3PW91 and BPW91) using 6‐311G(d,p) basis sets showed that McLafferty rearrangement of dibutyl, di(‐n‐octyl) and di(2‐ethyl‐n‐hexyl) phthalates is an energetically more favorable pathway than loss of an alcohol moiety. Prominent ions in these pathways were confirmed with deuterium labeled phthalates. Copyright © 2010 John Wiley & Sons, Ltd.     

Negative charge induced dissociation: fragmentation of deprotonated N‐benzylidene‐2‐hydroxylanilines in electrospray ionization mass spectrometry

Unimolecular reactivities of different N‐benzylidene‐2‐hydroxylaniline anions were investigated in gas phase by electrospray ionization tandem mass spectrometry. All the collision‐induced dissociation spectra of N‐benzylidene‐2‐hydroxylaniline anions show similar ions at phenyl anions, neutral loss of benzonitrile and benzoxazole anions, respectively. The possible fragmentation pathway was probed through deuterium labeling and various group substituents experiments. Computational results were applied to shed light on the mechanism of fragmentation patterns. The proton in the CH=N is reactive in the formation of the concerned ions. Its direct transfer to the oxygen results in 2‐hydroxyphenyl anion. Proton abstraction between benzoxazole and phenyl anion leads to the formation of benzene and benzoxazole anion. Copyright © 2014 John Wiley & Sons, Ltd.     

Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry

We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.     

Gas‐phase reactivity of 2‐hydroxy‐1,4‐naphthoquinones: a computational and mass spectrometry study of lapachol congeners

In order to understand the influence of alkyl side chains on the gas‐phase reactivity of 1,4‐naphthoquinone derivatives, some 2‐hydroxy‐1,4‐naphthoquinone derivatives have been prepared and studied by electrospray ionization tandem mass spectrometry in combination with computational quantum chemistry calculations. Protonation and deprotonation sites were suggested on the basis of gas‐phase basicity, proton affinity, gas‐phase acidity (ΔG_acid), atomic charges and frontier orbital analyses. The nature of the intramolecular interaction as well as of the hydrogen bond in the systems was investigated by the atoms‐in‐molecules theory and the natural bond orbital analysis. The results were compared with data published for lapachol (2‐hydroxy‐3‐(3‐methyl‐2‐butenyl)‐1,4‐naphthoquinone). For the protonated molecules, water elimination was verified to occur at lower proportion when compared with side chain elimination, as evidenced in earlier studies on lapachol. The side chain at position C(3) was found to play important roles in the fragmentation mechanisms of these compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

VUV photon‐induced ionization/dissociation of antipyrine and propyphenazone: mass spectrometric and theoretical insights

Two analgesic and anti‐inflammatory drugs, antipyrine and propyphenazone, were investigated with infrared laser desorption/tunable synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry (IR LD/VUV PIMS) and theoretical calculations. Mass spectra of the two drugs were measured at various photon energies. Fragment ions were gradually produced as photon energy increases. The structural assignment of the dominant fragment ions was supported by the results from a commercial electron impact time‐of‐flight mass spectrometer (EI‐TOF MS). Primary fragmentation pathways were established from experimental observations combining with theoretical calculations. Methyl radical elimination is a common fragmentation pathway for two analytes. However, for propyphenazone cation, isopropyl group elimination to form antipyrine cation is another competitive pathway. Copyright © 2010 John Wiley & Sons, Ltd.     

Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap-orbitrap mass spectrometry

The yeast Saccharomyces cerevisiae synthesizes three classes of sphingolipids: inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramides (MIPCs), and mannosyl-diinositolphosphoceramides (M(IP)2C). Tandem mass spectrometry of their molecular anions on a hybrid quadrupole time-of-flight (QqTOF) instrument produced fragments of inositol-containing head groups, which were specific for each lipid class. MS(n) analysis performed on a hybrid linear ion trap-orbitrap (LTQ Orbitrap) mass spectrometer with better than 3 ppm mass accuracy identified fragment ions specific for the amide-linked fatty acid and the long chain base moieties in individual molecular species. By selecting m/z of class-specific fragment ions for multiple precursor ion scanning, we profiled yeast sphingolipids in total lipid extracts on a QqTOF mass spectrometer. Thus, a combination of QqTOF and LTQ Orbitrap mass spectrometry lends itself to rapid, comprehensive and structure-specific profiling of the molecular composition of sphingolipids and glycerophospholipids in important model organisms, such as fungi and plants.     

Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray‐ tandem mass spectrometry

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI‐QTOF‐MS and direct infusion ESI‐FTICR‐MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision‐induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl‐ and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed‐phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI‐MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class. Copyright © 2008 John Wiley & Sons, Ltd.     

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.     

Nonvolatile salt‐free stabilizer for the quantification of polar imipenem and cilastatin in human plasma using hydrophilic interaction chromatography/quadrupole mass spectrometry with contamination sensitive off‐axis electrospray

A hydrophilic interaction chromatography/mass spectrometry (HILIC‐MS)‐based assay for imipenem (IMP) and cilastatin (CIL) was recently reported. This orthogonal electrospray ion source‐based (ORS) assay utilized nonvolatile salt (unremovable) to stabilize IMI in plasma. Unfortunately, this method was not applicable to conventional MS with off‐axis spray (OAS‐MS) because MS sensitivity was rapidly deteriorated by the nonvolatile salt. Therefore, we aimed to find a nonvolatile salt‐ and ion suppression‐free approach to stabilize and measure the analytes in plasma using OAS‐MS. Acetonitrile and methanol were tested to stabilize the analytes in the plasma samples. The recoveries, matrix effects and stabilities of the analytes in the stabilizer‐treated samples were studied. The variations in MS signal intensities were used as the indicator of the assay ruggedness. The results show that a mixture of methanol and acetonitrile (1:1) is best for the storage and measurement of IMP and CIL in human plasma. Utilization of this precipitant not only blocked the hydrolysis of the analytes in plasma but also resulted in an ion suppression‐free, fast (120 s per sample) and sensitive detection. The sensitivity obtained using the less sensitive OAS‐MS (API3000, 4 pg on column) is much greater than that of the published ORS‐MS‐based assay (API4000, 77 pg on column). The ruggedness of the assay was demonstrated by its constant MS signal intensity. In conclusion, an improved HILIC/MS‐based assay for IMP and CIL was established. The approach presented here provides a simple solution to the challenge of analyzing hydrolytically unstable β‐lactam antibiotics in biological samples. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Liquid chromatography–tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co‐eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H]⁺ ions at m/z at 833 and/or m/z 835, co‐eluted with uroporphyrin I and uroporphyrinogen I by LC‐MS/MS and could be wrongly identified as uroporphomethenes. Copyright © 2013 John Wiley & Sons, Ltd.     

The multiple conformational charge states of zinc(II) coordination by 2His‐2Cys oligopeptide investigated by ion mobility‐mass spectrometry,density functional theory and theoretical collision cross sections

Whether traveling wave ion mobility‐mass spectrometry (IM‐MS), B3LYP/LanL2DZ density functional theory, and ion size scaled Lennard‐Jones (LJ) collision cross sections (CCS) from the B3LYP optimized structures could be used to determine the type of Zn(II) coordination by the oligopeptide acetyl‐His₁‐Cys₂‐Gly₃‐Pro₄‐Tyr₅‐His₆‐Cys₇ (amb₅) was investigated. The IM‐MS analyses of a pH titration of molar equivalents of Zn(II):amb₅ showed that both negatively and positively charged complexes formed and coordination of Zn(II) increased as the His and Cys deprotonated near their pKa values. The B3LYP method was used to generate a series of alternative coordination structures to compare with the experimental results. The method predicted that the single negatively charged complex coordinated Zn(II) in a distorted tetrahedral geometry via the 2His‐2Cys substituent groups, whereas, the double negatively charged and positively charged complexes coordinated Zn(II) via His, carbonyl oxygens and the C‐terminus. The CCS of the B3LYP complexes were calculated using the LJ method and compared with those measured by IM‐MS for the various charge state complexes. The LJ method provided CCS that agreed with five of the alternative distorted tetrahedral and trigonal bipyramidal coordinations for the doubly charged complexes, but provided CCS that were 15 to 31 Ų larger than those measured by IM‐MS for the singly charged complexes. Collision‐induced dissociation of the Zn(II) complexes and a further pH titration study of amb_5B, which included amidation of the C‐terminus, suggested that the 2His‐2Cys coordination was more significant than coordinations that included the C‐terminus. Copyright © 2016 John Wiley & Sons, Ltd.     

Lithium adduct as precursor ion for sensitive and rapid quantification of 20 (S)‐protopanaxadiol in rat plasma by liquid chromatography/quadrupole linear ion trap mass spectrometry and application to rat pharmacokinetic study

A novel, rapid and sensitive liquid chromatography/quadrupole linear ion trap mass spectrometry [LC‐ESI‐(QqLIT)MS/MS] method was developed and validated for the quantification of protopanaxadiol (PPD) in rat plasma. Oleanolic acid (OA) was used as internal standard (IS). A simple protein precipitation based on acetonitrile (ACN) was employed. Chromatographic separation was performed on a Sepax GP‐C₁₈ column (50 × 2.1 mm, 5 μM) with a mobile phase consisting of ACN–water and 1.5 μM formic acid and 25 mM lithium acetate (90 : 10, v/v) at a flow rate of 0.4 ml/min for 3.0 min. Multiple‐reaction‐monitoring mode was performed using lithium adduct ion as precursor ion of m/z 467.5/449.4 and 455.6/407.4 for the drug and IS, respectively. Calibration curve was recovered over a concentration range of 0.5–100 ng/ml with a correlation coefficient >0.99. The limit of detection was 0.2 ng/ml in rat plasma for PPD. The results of the intraday and interday precision and accuracy studies were well within the acceptable limits. The validated method was successfully applied to investigate the pharmacokinetic study of PPD after intravenous and gavage administration to rat. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of β2‐agonists in human urine by fast‐gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 β₂‐agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid–liquid extraction, followed by derivatization of the extract and detection of β₂‐agonists trimethylsilyl‐derivatives by fast‐gas chromatography/electron impact–mass spectrometry (fast‐GC/EI‐MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra‐ and inter‐assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast‐GC/MS sequences, based on the use of short columns, high carrier‐gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS‐sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright © 2009 John Wiley & Sons, Ltd.