Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/ion trap mass spectrometry

An investigation of phosphate loss from phosphopeptide ions was conducted, using both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and electrospray ionization (ESI) coupled to an ion trap mass spectrometer (ITMS). These experiments were carried out on a number of phosphorylated peptides in order to investigate gas phase dephosphorylation patterns associated with phosphoserine, phosphothreonine, and phosphotyrosine residues. In particular, we explored the fragmentation patterns of phosphotyrosine containing peptides, which experience a loss of 98 Da under collision induced dissociation (CID) conditions in the ITMS. The loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. The fragmentation of phosphoserine and phosphothreonine containing peptides was also investigated. While phosphoserine and phosphothreonine residues undergo a loss of 98 Da under CID conditions regardless of peptide amino acid composition, phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence.     

Fragmentation studies of noncovalent sugar-sugar complexes by infrared atmospheric pressure MALDI

An investigation of sugar-sugar noncovalent complex fragmentation was conducted using a 2.94 microm Er:YAG laser for infrared (IR) atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) on an ion trap mass spectrometer (ITMS). This approach allowed the analysis of weak noncovalent complexes between a variety of biologically relevant oligosaccharides. The strength of interaction varied with different sugar structures, potentially due to varying strength of hydrogen bonding networks. In some cases, fragmentation of intramolecular sugar bonds preceded breakdown of the noncovalent complex. This result appeared primarily when complexes contained sugars with at least one sialic acid. Globotrios dimers also showed intramolecular fragmentation in preference to breakdown of the noncovalent dimer. This technique will allow further study of sugar-sugar interactions known to play a role in cellular interactions.     

Fragmentation study of hexanitrostilbene by ion trap multiple mass spectrometry and analysis by liquid chromatography/mass spectrometry

The fragmentation pathways of three explosive compounds with similar structures, hexanitrostilbene (HNS), cyclotrimethylene trinitramine (RDX), and 2,4,6-trinitrotoluene (TNT), have been investigated by multiple mass spectrometry (MSn, n = 1, 2, 3) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. The electron capture mechanism for these compounds in negative ion APCI and ESI mode differs from the usual negative ion mechanism, deprotonation or addition of other species. This was shown for HNS and TNT, which both gave a [M]- anion but not a [M-H]- ion in APCI, and the [M]- anion of HNS was observed in ESI. The quantitative analysis of HNS was performed by liquid chromatography (LC)/ESI-MS, and the results obtained by the internal standard (ISTD) method were compared with those from the external standard (ESTD) method, demonstrating that both quantitation approaches are useful, with good sensitivity, reproducibility and linearity, and ESTD is preferable in routine applications.     

Fragmentation study of protonated chalcones by atmospheric pressure chemical ionization and tandem mass spectrometry

Fragmentation mechanisms of protonated chalcone and its derivatives with different functional groups were investigated by atmospheric pressure chemical ionization with tandem mass spectrometry (MS/MS). The major fragmentation pathways were loss of the phenyl group from the A or B ring, combined with loss of CO. Losses of H(2)O and CO from the precursor ions of [M+H](+) are proposed to occur via rearrangements. Elimination of water from protonated chalcones was observed in all the title compounds to yield a stable ion but it was difficult to obtain skeletal fragmentation of a precursor ion. Loss of CO was found in the MS/MS spectra of all the compounds except the nitro-substituted chalcones. When the [M+H--CO](+) ion was fragmented in the MS/MS experiments, there were distinctive losses of 15 and 28 Da, as the methyl radical and ethylene, respectively. The ion at m/z 130, found only in the nitro-substituted chalcones, was assigned as C(9)H(6)O by Fourier transform ion cyclotron resonance (FTICR)-MS/MS; m/z 130 is a common fragment ion in the electron ionization (EI) spectra of chalcones. In order to more easily distinguish the constitutional isomers of these chalcones, breakdown curves were produced and these provided strong support in this study.     

Fragmentation studies of neutral per- and polyfluoroalkyl substances by atmospheric pressure ionization-multiple-stage mass spectrometry

The establishment of fragmentation pathways has a great interest in the identification of new or unknown related compounds present in complex samples. On that way, tentative fragmentation pathways for the ions generated by atmospheric pressure ionization of neutral per- and polyfluorinated alkyl substances (PFASs) have been proposed in this work. Electrospray (ESI), atmospheric pressure chemical ionization (APCI) and photoionization (APPI) were evaluated using mobile phases and source conditions that enhance the ionization efficiency of ions generated. A hybrid mass spectrometer consisting of a linear ion trap and an Orbitrap was used to combine the information of both multiple-stage mass spectrometry (MSⁿ) and mass accuracy measurements to characterize and establish the genealogical relationship between the product ions observed. The ionization mechanisms to generate ions such as [M–H]⁻, [M]^−•, and [M+O₂]^−• or the in-source collision-induced dissociation (CID) fragment ions in each API source are discussed in this study. In general, fluorotelomer olefins (FTOs) ionized in negative-ion APCI and APPI generated the molecular ion, while fluorotelomer alcohols (FTOHs) also provided the deprotonated molecule. Besides, fluorooctane sulfonamides (FOSAs) and sulfonamido-ethanols (FOSEs) led to the deprotonated molecule and in-source CID fragment ions, respectively. The fragmentation pathways from these precursor ions mainly involved initial α,β-eliminations of HF units and successive losses of CF₂ units coming from the perfluorinated alkyl chain. Moreover, FTOHs and FOSEs showed a high tendency to generate adduct ions under negative-ion ESI and APPI conditions. The fragmentation study of these adduct ions has demonstrated a strong interaction with the attached moiety.

Graphical abstract

     

Fragmentation induced in atmospheric pressure photoionization of peptides

In this work, the fragmentation of peptides under atmospheric pressure photoionization conditions is investigated. Intensive fragmentations into b/y- and c-sequence ions are reported. Abundance of these c-ions appeared to be related to the quantity of dopant infused and to the disappearance of the doubly protonated peptide ion. A careful analysis of the role of the dopant indicates that the fragmentations are not dependent on the nature of the dopant but on their ionization efficiencies. This result shows that the fragmentation arises from the reaction of the protonated peptide with photoelectrons released upon ionization of the dopant in an electron capture dissociation/electron transfer dissociation (ECD/ETD) type mechanism. Experiments with peptides bearing a single proton indicate that additional mechanisms are involved. H-atom transfer reactions are suggested to be responsible for the fragmentations as well. Those atoms could arise either from the dopant ions or from negatively charged solvent nanodroplets. This is the first report of an ECD/ETD mechanism in a dense medium and at atmospheric pressure.     

Ion trap mass spectrometry in the structural analysis of haemoglobin peptides modified by epichlorohydrin and diepoxybutane

Ion trap mass spectrometry has been shown to be particularly suitable for the structural analysis of high molecular weight peptides directly fragmented in the mass analyser without needing further sub-digestion reactions. Here we report the advantages of using multi-stage ion trap mass spectrometry in the structural characterisation of haemoglobin alkylated with epichlorohydrin and diepoxybutane. Alkylated globins were digested with trypsin and the peptide mixtures were analysed by MS(3). This technique allows the sequential fragmentation of peptides under analysis, giving rise to MS(3) product ion spectra with additional information with respect to MS(2) mass spectra. The results obtained complete the previously reported structural characterisation of alkylated haemoglobin, demonstrating the potential of ion trap mass spectrometry.     

Structural characterisation of unsaturated bacterial hopanoids by atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry

The production of bacteriohopanepolyols (BHPs) is widespread in many different groups of prokaryotes; however, unsaturated components are less common except amongst the acetic acid bacteria. Here we describe the characterisation of mono- (Delta(6) or Delta(11)) and diunsaturated (Delta(6,11)) bacteriohopanetetrols isolated from the acetic acid bacterium Gluconacetobacter xylinus (formerly Acetobacter aceti ssp. xylinum) by atmospheric pressure chemical ionisation ion trap mass spectrometry (APCI-MS(n)). APCI-MS(2) spectra are compared with equivalent electron ionisation (EI) spectra and differences in fragmentation pathways are discussed. Having established characteristic spectral features for a range of unsaturated BHPs we now have the ability to rapidly detect the presence of unsaturated BHPs in both natural environmental samples (soils, sediments, water columns) as well as in microbial cultures.     

Structural analysis of oligosaccharides by atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry.

An ion source incorporating a fibre optic interface has been constructed for atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry. The configuration has been applied to the study of linear and complex oligosaccharides. Multi-stage tandem mass spectrometry (MSn, n = 2-4) experiments carried out in the ion trap enable extended fragmentation pathways to be investigated that yield structural information. Collisional activation of sodiated oligosaccharides, as demonstrated on the model compound maltoheptaose, produces primarily B and Y fragments resulting from cleavage of glycosidic bonds; fragments from cross-ring cleavages are also observed following further stages of tandem mass spectrometry, providing additional linkage information. The analyses of mixtures of complex oligosaccharides are demonstrated for N-linked glycans from chicken egg glycoproteins and a ribonuclease glycan mixture. Mass spectrometric and tandem mass spectrometric data for sugars with molecular weights up to 4000 Da is shown for mixtures of linear dextrans and N-linked glycans. The use of MSn (n = 3, 4) on these complex molecules enabled structural information to be elucidated that confirms data observed in the MS/MS spectra.     

Analysis of peptides using partial (no discharge) atmospheric pressure chemical ionization conditions with ion trap mass spectrometry

A novel approach, based on the use of atmospheric pressure chemical ionization ion trap mass spectrometry (APCI-ITMS) conditions, but without using corona discharge, was used to analyze peptides. The proposed method was applied to three standard peptides (bombesin, trityrosine and tyrosine-glycine-glycine) as well as peptides obtained through enzymatic digestion of two standard proteins (horse cytochrome c and horse myoglobin).     

Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry

Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach.     

Characteristic fragmentation of bacteriohopanepolyols during atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry

Bacteriohopanepolyols (BHPs) fragment via characteristic pathways during atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry (APCI-LC/MS(n)). Comparison of the MS(2) spectra of bacteriohopane-32,33,34,35-tetrol (BHT) and 2 beta-methylbacteriohopane-32,33,34,35-tetrol has confirmed the previously proposed ring-C cleavage occurring between C-9 and 11 and C-8 and 14. This fragmentation, diagnostic of all hopanoids, also occurs in BHPs containing an amino group (-NH(2)) at C-35 although the higher relative stability of the ion limits this fragmentation to a minor process after protonation of the basic nitrogen function. Studies of a number of cell cultures including a prochlorophyte (Prochlorothrix hollandica) and a cyanobacterium (Chlorogloeopsis LA) demonstrate the power of this technique to detect composite BHPs with a complex biological functionality at C-35. We also report the first observation of intact pentafunctionalised bacteriohopanepolyols using this method.     

Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.     

Tandem mass spectrometry of alkali cationized polysaccharides in a quadrupole ion trap

Quadrupole ion trap mass spectrometry is used to study the linkage type dependent dissociation pathways of alkali-cationized disaccharides, mostly of the type glucosyl(1 → X)glucose (X = 1, 2, 3, 4, or 6). The reaction mechanisms of a set of disaccharides containing all possible α anomeric linkage types and some β anomers are probed with tandem mass spectrometry, MS ⁿ , and double resonance experiments. Tandem mass spectrometry experiments on an ¹⁸O-labeled disaccharide show that the dissociation paths for Li and Na cationized species are the same. Experiments on three trisaccharides (isomaltotriose, maltotriose, and panose), a tetrasaccharide (isomaltotetraose), and a pentasaccharide (maltopentaose) show that tandem mass spectrometry provides all available linkage information and MS ⁿ can provide selected linkage information. The mode of alkali binding is examined via semiempirical calculations and by measuring alkali-carbohydrate relative cation affinities.     

Rapid structural elucidation of composite bacterial hopanoids by atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry

Bacteriohopanepolyols (BHPs) are membrane lipids produced by a wide range of eubacteria. Their use, however, as molecular markers of bacterial populations and processes has until recently been hampered by the lack of a suitable rapid method for fingerprinting their composition in complex environmental matrices. New analytical procedures employing ion trap mass spectrometry now allow us to investigate the occurrence of BHPs in diverse biological and environmental samples including bacterial cultures, soils, and recent and ancient sediments. Here, we describe the structural characterisation using atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry (APCI-LC/MS(n)) of a number of previously identified but less commonly occurring BHPs such as adenosylhopane and ribonylhopane. Many of the structures described here have previously only been reported in one or just a small number of cultured organisms having been isolated from large amounts of cellular mass (4-26 g) and identified by nuclear magnetic resonance (NMR) techniques after purification of individual compounds. Now, having established characteristic APCI fragmentation patterns, it is possible to rapidly screen many more bacterial cultures using only small amounts of material (<50 mg) as well as environmental samples for these atypical structures and a rapidly growing suite of novel structures.     

Ion trap mass spectrometry: a personal perspective

This paper is a personal perspective of the commercial development of the three-dimensional quadrupole ion trap mass spectrometer. Early ion trap invention and development which dates back to 1953, is described. The development of the ion trap is traced through three ages with the last age being where commercial development takes place. Key technical breakthroughs in ion trap technology and commercialization are presented and described up to the present time.     

Fragmentation investigation of brassinosteroid compounds by ion trap and quadrupole time-of-flight mass spectrometry

The fragmentation mechanisms of three types of brassinosteroids (BRs), 23,24‐tris‐epicastasterone, epicastasterone, tris–epicastasterone, 24‐epibrassinolide and 6‐deoxo‐24‐epicastasterone, have been extensively investigated by tandem mass spectrometry (MSⁿ, n = 1, 2, 3, 4, 5) with the assistance of high mass accuracy quadrupole time‐of‐flight mass spectrometry (QToF MS). The electrospray ionization (ESI) mass spectrometric fragmentation pathways of these five BRs were comprehensively elucidated for the first time. Cleavages of side chains, neutral losses of water or other molecules and opening of a ring induce the main fragmentation patterns. The results from the present study can potentially afford important guidance for the structural elucidation of different BRs and provide some fundamental data for metabolomic analysis of BRs. Copyright © 2010 John Wiley & Sons, Ltd.     

大气压离子化技术/飞行时间质谱联用进展

综述了大气压离子化技术/飞行时间质谱联用技术及其应用的进展     

Inline pneumatically assisted atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry

Atmospheric pressure (AP) matrix‐assisted laser desorption/ionization (MALDI) is known to suffer from poor ion transfer efficiencies as compared to conventional vacuum MALDI (vMALDI). To mitigate these issues, a new AP‐MALDI ion source utilizing a coaxial gas flow was developed. Nitrogen, helium, and sulfur hexafluoride were tested for their abilities as ion carriers for a standard peptide and small drug molecules. Nitrogen showed the best ion transport efficiency, with sensitivity gains of up to 1900% and 20% for a peptide standard when the target plate voltage was either continuous or pulsed, respectively. The addition of carrier gas not only entrained the ions efficiently but also deflected background species and declustered analyte–matrix adducts, resulting in higher absolute analyte signal intensities and greater signal‐to‐noise (S/N) ratios. With the increased sensitivity of pneumatically assisted (PA) AP‐MALDI, the limits of detection of angiotensin I were 20 or 3 fmols for continuous or pulsed target plate voltage, respectively. For analyzing low‐mass analytes, it was found that very low gas flow rates (0.3–0.6 l min^?1) were preferable owing to increased fragmentation at higher gas flows. The analyte lability, type of gas, and nature of the extraction field between the target plate and mass spectrometer inlet were observed to be the most important factors affecting the performance of the in‐line PA‐AP‐MALDI ion source. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterisation of sulphoxides by atmospheric pressure ionisation mass spectrometry

An observation that a series of proprietary compounds containing a methyl thiophenyl group all underwent metabolic S-oxidation, and that the product ion spectra of the resulting S-oxides showed methyl radical loss under low-energy atmospheric pressure ionisation tandem mass spectrometry (API-MS/MS) conditions, has led to an investigation of the fragmentation of commercially available sulphoxides. The phenyl methyl sulphoxides studied do lose methyl radicals under MS/MS conditions on triple quadrupole mass spectrometers. In addition, the phenyl sulphoxides, with simple substituents other than a methyl group, also showed a tendency to lose the substituent as a radical. It was concluded that radical loss from these simple sulphoxides was characteristic of S-oxidation of these molecules. Radical losses, such as those reported here, are used in-house to distinguish S-oxidation from N- and C-oxidation in metabolism studies.