The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Analysis of antioxidants in insulation cladding of copper wire: a comparison of different mass spectrometric techniques (ESI–IT,MALDI–RTOF and RTOF–SIMS)

Commercial copper wire and its polymer insulation cladding was investigated for the presence of three synthetic antioxidants (ADK STAB AO412S, Irganox 1010 and Irganox MD 1024) by three different mass spectrometric techniques including electrospray ionization–ion trap–mass spectrometry (ESI–IT–MS), matrix‐assisted laser desorption/ionization reflectron time‐of‐flight (TOF) mass spectrometry (MALDI–RTOF–MS) and reflectron TOF secondary ion mass spectrometry (RTOF–SIMS). The samples were analyzed either directly without any treatment (RTOF–SIMS) or after a simple liquid/liquid extraction step (ESI–IT–MS, MALDI–RTOF–MS and RTOF–SIMS). Direct analysis of the copper wire itself or of the insulation cladding by RTOF–SIMS allowed the detection of at least two of the three antioxidants but at rather low sensitivity as molecular radical cations and with fairly strong fragmentation (due to the highly energetic ion beam of the primary ion gun). ESI–IT‐ and MALDI–RTOF–MS‐generated abundant protonated and/or cationized molecules (ammoniated or sodiated) from the liquid/liquid extract. Only ESI–IT–MS allowed simultaneous detection of all three analytes in the extract of insulation claddings. The latter two so‐called ‘soft’ desorption/ionization techniques exhibited intense fragmentation only by applying low‐energy collision‐induced dissociation (CID) tandem MS on a multistage ion trap‐instrument and high‐energy CID on a tandem TOF‐instrument (TOF/RTOF), respectively. Strong differences in the fragmentation behavior of the three analytes could be observed between the different CID spectra obtained from either the IT‐instrument (collision energy in the very low eV range) or the TOF/RTOF‐instrument (collision energy 20 keV), but both delivered important structural information. Copyright © 2009 John Wiley & Sons, Ltd.     

The fragmentation of ethoxylated surfactants by AP-MALDI-QIT

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important technique to characterize the chemical structure of industrial polymer materials. MALDI methods have been developed to address a broad variety of different polymer materials containing different chemistries. One of the key aspects of the typical MALDI experiment is the generation of intact ions. The development of Atmospheric Pressure (AP) MALDI quadrupole ion trap (QIT) instruments has opened another channel to obtaining MS/MS experiments for polymer samples. These experiments provide a new method to obtain chemical structure information from MALDI experiments. Collision-Induced Dissociation (CID) provides an improved MALDI MS/MS experiment that can be done on readily available mass spectrometers. AP MALDI QIT techniques have been successfully applied to a variety of synthetic polymers. This work explores the applicability of AP MALDI QIT methods to relatively low molecular weight ethoxylated surfactants. In these experiments we show the CID fragmentation mass spectra on some ethoxylated surfactants, and demonstrate the existence of analyte matrix clusters.     

Structural characterization of polyethers using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Fragmentation of polyethers, such as poly(ethylene glycol) (PEG), poly(propylene glycol) and poly(tetramethylene glycol) was analyzed by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) using a quadrupole ion trap time-of-flight mass spectrometer (QIT-ToF). The Li adduct ion provided more abundant fragments than the Na and K adduct ions in the MS/MS spectra. A previous study had demonstrated four series fragments of hydroxyl-, vinyl- and formyl-terminated ions, as well as distonic cations, in high-energy fast atom bombardment MS/MS and MALDI collision-induced dissociation measurements of poly(ethylene glycol). In the present study, the low-energy MS/MS measurements using MALDI-QIT-ToF, showed hydroxyl-, vinyl- and formyl- terminated fragments with or without other fragment groups, but not distonic cations. The fragmentation depended on the types of polyethers examined. MS/MS measurements using MALDI-QIT-ToF are expected to allow structural characterization of unknown components of polyethers.     

Negative and positive ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and positive ion nano-electrospray ionization quadrupole ion trap mass spectrometry of peptidoglycan fragments isolated from various Bacillus species

A general approach for the detailed characterization of sodium borohydride-reduced peptidoglycan fragments (syn. muropeptides), produced by muramidase digestion of the purified sacculus isolated from Bacillus subtilis (vegetative cell form of the wild type and a dacA mutant) and Bacillus megaterium (endospore form), is outlined based on UV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS). After enzymatic digestion and reduction of the resulting muropeptides, the complex glycopeptide mixture was separated and fractionated by reversed-phase high-performance liquid chromatography. Prior to mass spectrometric analysis, the muropeptide samples were subjected to a desalting step and an aliquot was taken for amino acid analysis. Initial molecular mass determination of these peptidoglycan fragments (ranging from monomeric to tetrameric muropeptides) was performed by positive and negative ion MALDI-MS using the thin-layer technique with the matrix alpha-cyano-4-hydroxycinnamic acid. The results demonstrated that for the fast molecular mass determination of large sample numbers in the 0.8-10 pmol range and with a mass accuracy of +/-0.07%, negative ion MALDI-MS in the linear TOF mode is the method of choice. After this kind of muropeptide screening often a detailed primary structural analysis is required owing to ambiguous data. Structural data could be obtained from peptidoglycan monomers by post-source decay (PSD) fragment ion analysis, but not from dimers or higher oligomers and not with the necessary sensitivity. Multistage collision-induced dissociation (CID) experiments performed on an nESI-QIT instrument were found to be the superior method for structural characterization of not only monomeric but also of dimeric and trimeric muropeptides. Up to MS4 experiments were sometimes necessary to obtain unambiguous structural information. Three examples are presented: (a) CID MSn (n = 2-4) of a peptidoglycan monomer (disaccharide-tripeptide) isolated from B. subtilis (wild type, vegetative cell form), (b) CID MSn (n = 2-4) of a peptidoglycan dimer (bis-disaccharide-tetrapentapeptide) obtained from a B. subtilis mutant (vegetative cell form) and (c) CID MS2 of a peptidoglycan trimer (a linear hexasaccharide with two peptide side chains) isolated from the spore cortex of B. megaterium. All MS(n) experiments were performed on singly charged precursor ions and the MS2 spectra were dominated by fragments derived from interglycosidic bond cleavages. MS3 and MS4 spectra exhibited mainly peptide moiety fragment ions. In case of the bis-disaccharide-tetrapentapeptide, the peptide branching point could be determined based on MS3 and MS4 spectra. The results demonstrate the utility of nESI-QIT-MS towards the facile determination of the glycan sequence, the peptide linkage and the peptide sequence and branching of purified muropeptides (monomeric up to trimeric forms). The wealth of structural information generated by nESI-QIT-MSn is unsurpassed by any other individual technique.     

基质辅助激光解吸电离质谱和电喷雾电离质谱在辣根过氧化物酶糖肽结构分析中的应用

糖链结构的质谱解析是今后糖蛋白分析中的重要研究内容,其中完整糖肽的分析,由于可以同时获得糖基化位点和对应糖链的结构信息,更具有重要意义和研究前景。本工作对质谱软电离技术在完整糖肽分析中的应用进行了研究,其中包括了基质辅助激光解吸电离(matrix-assisted laser desorption ionization, MALDI)和电喷雾电离(electrospray ionization, ESI)技术。通过平行使用两种串联质谱(tandem mass spectrometry, MS/MS)分析策略: MALDI-MS/MS和ESI-MS/MS对目标糖蛋白——辣根过氧化物酶进行分析,并讨论了其互补性。结果表明,MALDI和ESI技术各有优劣,结合串联质谱分析,可获得糖肽的糖链结构信息;两条路线互补使用,在揭示蛋白质糖基化修饰(位点和结构)的研究中十分必要。     

Electron capture dissociation of polypeptides in a three-dimensional quadrupole ion trap: Implementation and first results

The adverse influence of the radio frequency (RF) voltage on electrons has been the main obstacle for the implementation of electron capture dissociation (ECD) in three-dimensional quadrupole ion traps (3D QITs). Here we demonstrate that the use of axial magnetic field, together with the injection of low-energy (<5 eV) electrons, in the beginning of the positive RF semi-period achieves trapping of electrons for a period of time comparable with the semi-period duration. Importantly, the energy of the electrons remains low during most of the trapping period. With this technique, which we call "magnetized electrons, in-phase injection" (MEPhI), ECD and other ion-electron reactions have become possible in a 3D QIT. Initial ECD results, including single-scan data, were obtained with dications of Substance P. The observed secondary fragmentation of ECD fragments indicates that the trapped electrons are still somewhat hotter than desired.     

Analysis of a chlorosulfolipid from Ochromonas danica by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) is an established tool for analyzing high mass molecules, such as proteins, whereas it attracts far less interest in the field of lipid analysis. In the study reported here a new chlorosulfolipid (CSL), 3,8,12,15-tetrachloroeicosane-1,17,18-triyl tris(hydrogen sulfate), was identified from the alga Ochromonas danica and de novo characterized by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-ToF) MS in negative ion mode. This method provides an effective alternative for the analysis of compounds directly derived from organic cell extracts. For MALDI analyses several frequently used solid MALDI matrices as well as some ionic liquid matrices (ILMs) were tested to enhance the analyte response to UV-laser and its ionization. The molecular weight of the observed compound could be determined as Li-, Na- and K-adducts [M+Me-2H]-. The characteristic isotopic patterns of the measured ions and the well-allocated molecular fragments by MS1, MS2 and MS3 indicate the fourfold chlorination and threefold sulfation of the investigated compound. The MS fragmentation alongside of the chlorine-bearing C-atoms is accompanied by the generation of a double bond at the opposite fragment in MS1. This obtained fragmentation pattern provides an insight into the allocation of the chlorine-bearing C-atoms along the carbon chain.     

Analysis of high mass peptides using a novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometer

A novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight (MALDI QIT ToF) mass spectrometer has been used to analyse high mass peptide ions exceeding 2000 Da. Human adrenocorticotropic hormone (fragment 18-39) and oxidised bovine insulin chain B were utilised to evaluate the performance of the instrument both in MS and in MS/MS mode. Its ability to efficiently isolate ions and to fragment them using collisionally activated decomposition (CAD) has been demonstrated using mixtures diluted to the low-femtomole level on target. Additionally, multiple stage mass spectrometry (MS/MS/MS) provides a second-generation product ion spectrum in which new fragment ions are detected and new stretches of amino acids are identified.     

Detection of pharmaceutical compounds in tissue by matrix-assisted laser desorption/ionization and laser desorption/chemical ionization tandem mass spectrometry with a quadrupole ion trap

A novel quadrupole ion trap mass spectrometer laser microprobe instrument with an external ionization source was constructed and used to investigate the matrix-assisted laser desorption/ionization (MALDI) detection of pharmaceutical compounds in intact tissue. In addition to MALDI, laser desorption coupled with chemical ionization (LD/CI) was investigated. MALDI, using 2,5-dihydroxybenezoic acid (DHB) as a matrix, was employed to detect the anticancer drug paclitaxel from a thin section of rat liver tissue which had been incubated in a solution of paclitaxel. The results of that experiment showed that the ability to perform tandem mass spectrometry (MS/MS) with the quadrupole ion trap was crucial in the identification of drug compounds at trace levels in the complex tissue matrix. MALDI MS/MS was then used to detect the presence of paclitaxel in a human ovarian tumor at a concentration of approximately 50 mg/kg. Finally, the drug spiperone was detected in incubated rat liver tissue at an approximate level of 25 mg/kg using LD/CI (no MALDI matrix). Again, the MS/MS capability of the quadrupole ion trap was crucial in the identification of the drug at trace levels in the complex tissue matrix.     

A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance

A matrix-assisted laser desorption/ionization (MALDI) source has been coupled to a tandem quadrupole/time-of-flight (QqTOF) mass spectrometer by means of a collisional damping interface. Mass resolving power of about 10,000 (FWHM) and accuracy in the range of 10 ppm are observed in both single-MS mode and MS/MS mode. Sub-femtomole sensitivity is obtained in single-MS mode, and a few femtomoles in MS/MS mode. Both peptide mass mapping and collision-induced dissociation (CID) analysis of tryptic peptides can be performed from the same MALDI target. Rapid spectral acquisition (a few seconds per spectrum) can be achieved in both modes, so high throughput protein identification is possible. Some information about fragmentation patterns was obtained from a study of the CID spectra of singly charged peptides from a tryptic digest of E. coli citrate synthase. Reasonably successful automatic sequence prediction (>90%) is possible from the CID spectra of singly charged peptides using the SCIEX Predict Sequence routine. Ion production at pressures near 1 Torr (rather than in vacuum) is found to give reduced metastable fragmentation, particularly for higher mass molecular ions. Copyright 2000 John Wiley & Sons, Ltd.     

Dissociation of biomolecules using a ultraviolet matrix-assisted laser desorption/ionisation time-of-flight/curved field reflectron tandem mass spectrometer equipped with a differential-pumped collision cell

A commercial matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) instrument equipped with a curved field reflectron (CFR) was modified in order to perform collision-induced dissociation (CID) on a variety of biomolecules. The incorporation of a high-resolution ion gate together with a collision cell within the field-free region allowed tandem mass analysis (MS/MS), without the necessity to decelerate the precursor ions prior to activation. The simultaneous detection of all product ions remained possible by using the CFR. To test the MS/MS performances, ACTH (fragment 1-17), a complex high mannose carbohydrate (Man)(8)(GlcNac)(2) and a lysophosphatidylcholine lipid (18:1) were analysed on the modified instrument. Direct comparison with the low-energy product ion spectra, acquired on a MALDI quadrupole ion trap (QIT) two-stage reflectron time-of flight (ReToF) mass spectrometer, showed significant differences in the types of product ions observed. The additional ions detected were a clear indication of the high-energy fragmentation processes occurring in the collision cell.     

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.     

Quantitative aspects in electrospray ionization ion trap and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of malto-oligosaccharides

Mass spectrometry is widely applied in carbohydrate analysis, but still quantitative evaluation of data is critical due to different ionization efficiencies of the constituents in a mixture. Different size and chemical structure of the analytes cause their uneven distribution in droplets (electrospray ionization, ESI) or matrix spots (matrix-assisted laser desorption/ionization, MALDI). In addition, instrumental parameters affect final ion yields. In order to study and optimize the latter, an equimolar mixture of malto-oligosaccharides (DP1-6) was analyzed using varying target masses for ESI as well as different matrices and laser power for MALDI. The sodium adducts and derivatives for positive ion mode (hydrazones with Girard's T Reagent, GT) and negative ion mode (reductively aminated with o-aminobenzoic acid, oABA) were studied. Negatively charged oABA-labeled malto-oligosaccharides turned out to be unsuitable for quantification of the malto-oligomeric composition. Best agreement was achieved when applying target masses in the range of the highest homolog in the mixture in electrospray ionization ion trap (ESI-IT) (1-2% deviation with GT label or as Na(+) adducts). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) gave best results when the laser power was adjusted significantly over the desorption/ionization threshold (1% deviation with GT label). Both parameters show significant influence on the determined oligomeric composition. Consequently, estimation and even quantitative determination of amounts of oligosaccharides in a mixture can be achieved when the analytes are labeled and the proper instrumental parameters are used.     

Comparison of CID spectra of singly charged polypeptide antibiotic precursor ions obtained by positive-ion vacuum MALDI IT/RTOF and TOF/RTOF, AP-MALDI-IT and ESI-IT mass spectrometry

Various classes of polypeptide antibiotics, including blocked linear peptides (gramicidin D), side-chain-cyclized peptides (bacitracin, viomycin, capreomycin), side-chain-cyclized depsipeptides (virginiamycin S), real cyclic peptides (tyrocidin, gramcidin S) and side-chain-cyclized lipopeptides (polymyxin B and E, amfomycin), were investigated by low-energy collision induced dissociation (LE-CID) as well as high-energy CID (HE-CID). Ion trap (IT) based instruments with different desorption/ionization techniques such as electrospray ionization (ESI), atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and vacuum MALDI (vMALDI) as well as a vMALDI-time-of-flight (TOF)/curved field-reflectron instrument fitted with a gas collision cell were used. For optimum comparability of data from different IT instruments, the CID conditions were standardized and only singly charged precursor ions were considered. Additionally, HE-CID data obtained from the TOF-based instrument were acquired and compared with LE-CID data from ITs. Major differences between trap-based and TOF-based CID data are that the latter data set lacks abundant additional loss of small neutrals (e.g. ammonia, water) but contains product ions down to the immonium-ion-type region, thereby allowing the detection of even single amino-acid (even unusual amino acids) substitutions. For several polypeptide antibiotics, mass spectrometric as well as tandem mass spectrometric data are shown and discussed for the first time, and some yet undescribed minor components are also reported. De novo sequencing of unusually linked minor components of (e.g. cyclic) polypeptides is practically impossible without knowledge of the exact structure and fragmentation behavior of the major components. Finally, the described standardized CID condition constitutes a basic prerequisite for creating a searchable, annotated MS(n)-database of bioactive compounds. The applied desorption/ionization techniques showed no significant influence on the type of product ions (neglecting relative abundances of product ions formed) observed, and therefore the type of analyzer connected with the CID process mainly determines the type of fragment ions.     

Dynamic collision-induced dissociation (DCID) in a quadrupole ion trap using a two-frequency excitation waveform: I. Effects of excitation frequency and phase angle

This study describes the application of a two-frequency excitation waveform to the end-cap electrodes of a quadrupole ion trap (QIT) during the mass acquisition period to deliberately fragment selected precursor ions. This approach obviates the need for a discrete excitation period and guarantees on-resonant excitation conditions without any requirement for resonant tuning; it is therefore faster than the conventional approach to collision-induced dissociation (CID) in QITs. The molecular ion of n-butylbenzene is used as thermometer molecule to determine the energetics of the new excitation procedure. The excitation waveform, consisting of two closely spaced sinusoidal frequencies, has an interference pattern that displays nodes and crests in the time domain. The energetics (determined by the product ion ratios of 91/92 Th) and CID efficiencies are highly dependent on the excitation amplitude, the relative position of the excitation frequencies in the Mathieu stability diagram, and whether the ions come into resonance during a node or crest of the excitation waveform. Under highly energetic conditions, ratios of 91/92 as large as 15 can be obtained at concomitant CID efficiencies of 10%, indicating internal energies in excess of 10 eV at the time of fragmentation. These extremely high internal energies far exceed the energetics achievable using conventional on-resonance excitation in QITs, indicating that the collisional heating rate is very fast in the new approach. Under less energetic conditions CID efficiencies as high as 70% are possible, which compares favorably with results obtained by conventional on-resonance excitation. Correlation analyses are used to determine the conditions that simultaneously optimize energetic and efficient fragmentation conditions.     

Elimination of isocyanate and isothiocyanate molecules at the electrospray ionization ion trap, electrospray ionization quadrupole time-of-flight and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry fragmentation of sodium cationized brassitin, brassinin and their glycosides

Fragmentation mechanisms of phytoalexin analogs, including brassitin and brassinin and their glucosylated analogs, have been studied by electrospray (ESI) ion trap (IT) multistage (MS(n), n = 1-4) mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF/ToF) and ESI-Q/ToF tandem mass spectrometry techniques. At the fragmentation of sodium adducts a hitherto not described process has been elucidated The proposed mechanism of this process includes cyclization of the brassitin and brassinin cationized adducts through a six-membered cycle of the molecules and the elimination of isocyanate or isothiocyanate from the thio- or dithiocarbamate moiety, giving rise to [M + Na - 43](+) or [M + Na - 59](+) adducts. The elimination of NH=C=O or NH=C=S molecules has been confirmed by the high resolution measurement of the elemental composition of the ions produced and quantum-chemical calculations of the six-membered transition state. Other fragmentation routes include cleavage of an alkane linker, while numerous characteristic hexopyranose pathways are taking place in the glucosylated compounds. The presented theoretical data on the ESI and MALDI behavior of the saccharidic, as well as of the indole aglycon parts, can facilitate structural elucidation of the analogous compounds.     

Rapid identification of gallotannins from Chinese galls by matrix‐assisted laser desorption/ionization time‐of‐flight quadrupole ion trap mass spectrometry

Chinese gall, a conventional traditional Chinese medicine, contains high levels of gallotannins. A rapid method for direct analysis of the gallotannins without using any troublesome sample pretreatments was developed using matrix‐assisted laser desorption/ionization time‐of‐flight quadrupole ion trap mass spectrometry (MALDI‐QIT‐TOF MS) to successfully identify the gallotannin components in the crude extract of Chinese galls within several minutes. The high quality of the MS and MS² spectra acquired clearly showed that hydrolysable tannins in Chinese galls were identified as a series of the gallotannins with degrees of polymerization (DP) of 4–11 galloyl units. The MS² data indicated that the identified gallotannins with DP of 4–7 galloyl units had clear fragmentation with loss of 1–5 galloyl units which were further deprived of 1–3 water moieties. This technique may be used for rapid evaluation and screening of hydrolysable tannins in medicinal plants. Copyright © 2009 John Wiley & Sons, Ltd.     

On the time scale of internal energy relaxation of AP-MALDI and nano-ESI Ions in a quadrupole ion trap

Recently reported results (Konn et al. [14]) on the collisional cooling of atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) and nano-electrospray ionization (nano-ESI) generated ions in a quadrupole ion trap mass spectrometer (QITMS) are inconsistent with measured collisional cooling rates. The work reported here presents a re-examination of those previous results. Collision induced dissociation (CID) has been used to probe various properties of ions contained in a QITMS. It is shown experimentally that when trapping large numbers of ions, an effective dc trapping voltage is induced that varies with changes in the size of the ion cloud. A decrease in the resonant frequency for maximum CID efficiency is observed as the cool time between parent ion isolation and CID is increased. Ion trajectories in a QITMS are simulated to demonstrate how ion density changes over the course of parent ion isolation. The effect of space charge on ion motion is simulated, and Fourier transformations of ion axial motion plus simple calculations corroborate the experimentally observed transient frequency shifts. The relative stability of ions formed by AP-MALDI and nano-ESI is compared under low charge density conditions. These data show that the ions have reached equilibrium internal energy and, thus, that differences in dissociation onsets and “50% fragmentation efficiency points” between the ionization mechanisms are due to the formation of distinct ion conformations as previously shown in reference [28]. The conclusions of Konn et al. [14] are based on invalid experimental procedures as well as inappropriate comparisons of QITMS data to low-pressure FT-ICR data.     

Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Protein identification is routinely accomplished by peptide sequencing using mass spectrometry (MS) after enzymatic digestion. Site-specific chemical modification may improve peptide ionization efficiency or sequence coverage in mass spectrometry. We report herein that amino group of lysine residue in peptides can be selectively modified by reaction with a peroxycarbonate and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID) in electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS. Selective modification of lysine residue in peptides by our strategy can induce specific peptide cleavage at or near the lysine site. Studies using deuterated analogues of modified lysine indicate that fragmentation of the modified peptides involves apparent free-radical processes that lead to peptide chain fragmentation and side-chain loss. The formation of a-, c-, or z-types of ions in MS is reminiscent of the proposed free-radical mechanisms in low-energy electron capture dissociation (ECD) processes that may have better sequence coverage than that of the conventional CID method. This site-specific cleavage of peptides by free radical- promoted processes is feasible and such strategies may aid the protein sequencing analysis and have potential applications in top-down proteomics.