High-accuracy MS/MS spectra of deprotonated ions of 390 dipeptides and 137 peptides with three to six residues are studied. Many amino acid residues undergo neutral losses from their side chains. The most abundant is the loss of acetaldehyde from threonine. The abundance of losses from the side chains of other amino acids is estimated relative to that of threonine. While some amino acids lose the whole side chain, others lose only part of it, and some exhibit two or more different losses. Side-chain neutral losses are less abundant in the spectra of protonated peptides, being significant mainly for methionine and arginine. In addition to the neutral losses, many amino acid residues in deprotonated peptides produce specific negative ions after peptide bond cleavage. An expanded list of fragment ions from protonated peptides is also presented and compared with those of deprotonated peptides. Fragment ions are mostly different for these two cases. These lists of fragments are used to annotate peptide mass spectral libraries and to aid in the confirmation of specific amino acids in peptides.
Ion mobility spectrometry (IMS) coupled with gas-phase hydrogen deuterium exchange (HDX)-mass spectrometry (MS) and molecular dynamic simulations (MDS) has been used for structural investigation of anions produced by electrospraying a sample containing a synthetic peptide having the sequence KKDDDDDIIKIIK. In these experiments the potential of the analytical method for locating charge sites on ions as well as for utilizing collision-induced dissociation (CID) to reveal the degree of deuterium uptake within specific amino acid residues has been assessed. For diffuse (i.e., more elongated) [M – 2H]2– ions, decreased deuterium content along with MDS data suggest that the D4 and D6 residues are charge sites, whereas for the more diffuse [M – 3H]3– ions, the data suggest that the D4, D7, and the C-terminus are deprotonated. Fragmentation of mobility-selected, diffuse [M – 2H]2– ions to determine deuterium uptake at individual amino acid residues reveals a degree of deuterium retention at incorporation sites. Although the diffuse [M – 3H]3– ions may show more HD scrambling, it is not possible to clearly distinguish HD scrambling from the expected deuterium uptake based on a hydrogen accessibility model. The capability of the IMS-HDX-MS/MS approach to provide relevant details about ion structure is discussed. Additionally, the ability to extend the approach for locating protonation sites on positively-charged ions is presented.
Peptide cation-radical fragment ions of the z-type, [●AXAR+], [●AXAK+], and [●XAR+], where X = A, C, D, E, F, G, H, K, L, M, N, P, Y, and W, were generated by electron transfer dissociation of peptide dications and investigated by MS3-near-ultraviolet photodissociation (UVPD) at 355 nm. Laser-pulse dependence measurements indicated that the ion populations were homogeneous for most X residues except phenylalanine. UVPD resulted in dissociations of backbone CO─NH bonds that were accompanied by hydrogen atom transfer, producing fragment ions of the [yn]+ type. Compared with collision-induced dissociation, UVPD yielded less side-chain dissociations even for residues that are sensitive to radical-induced side-chain bond cleavages. The backbone dissociations are triggered by transitions to second (B) excited electronic states in the peptide ion R-CH●-CONH- chromophores that are resonant with the 355-nm photon energy. Electron promotion increases the polarity of the B excited states, R-CH+-C●(O–)NH-, and steers the reaction to proceed by transfer of protons from proximate acidic Cα and amide nitrogen positions.
Carboxamides bearing an N–H functionality are known to undergo deprotonation under negative-ion-generating mass spectrometric conditions. Herein, we report that N–H bearing carboxamides with acidities lower than that of the hydroperoxyl radical (HO-O?) preferentially form superoxide radical-anion (O2-?) adducts, rather than deprotonate, when they are exposed to the glow discharge of a helium-plasma ionization source. For example, the spectra of N-alkylacetamides show peaks for superoxide radical-anion (O2-?) adducts. Conversely, more acidic amides, such as N-alkyltrifluoroacetamides, preferentially undergo deprotonation under similar experimental conditions. Upon collisional activation, the O2-? adducts of N-alkylacetamides either lose the neutral amide or the hydroperoxyl radical (HO-O?) to generate the superoxide radical-anion (m/z 32) or the deprotonated amide [m/z (M – H)?], respectively. For somewhat acidic carboxamides, the association between the two entities is weak. Thus, upon mildest collisional activation, the adduct dissociates to eject the superoxide anion. Superoxide-adduct formation results are useful for structure determination purposes because carboxamides devoid of a N–H functionality undergo neither deprotonation nor adduct formation under HePI conditions.
Changes in protein ion conformation as a result of nonspecific adduction of metal ions to the protein during electrospray ionization (ESI) from aqueous solutions were investigated using traveling wave ion mobility spectrometry (TWIMS). For all proteins examined, protein cations (and in most cases anions) with nonspecific metal ion adducts are more compact than the fully protonated (or deprotonated) ions with the same charge state. Compaction of protein cations upon nonspecific metal ion binding is most significant for intermediate charge state ions, and there is a greater reduction in collisional cross section with increasing number of metal ion adducts and increasing ion valency, consistent with an electrostatic interaction between the ions and the protein. Protein cations with the greatest number of adducted metal ions are no more compact than the lowest protonated ions formed from aqueous solutions. These results show that smaller collisional cross sections for metal-attached protein ions are not a good indicator of a specific metal–protein interaction in solution because nonspecific metal ion adduction also results in smaller gaseous protein cation cross sections. In contrast, the collisional cross section of α-lactalbumin, which specifically binds one Ca2+, is larger for the holo-form compared with the apo-form, in agreement with solution-phase measurements. Because compaction of protein cations occurs when metal ion adduction is nonspecific, elongation of a protein cation may be a more reliable indicator that a specific metal ion–protein interaction occurs in solution. 相似文献
In negative electrospray ionization mass spectrometry of 4-nitrobenzyl 4-hydroxybenzoates, a decarboxylation reaction, which was significantly promoted by the presence of a nitro group on the benzyl group, competed with radical elimination reactions. Density functional theory calculations indicated that decarboxylation of deprotonated 4-nitrobenzyl vanillate occurred via a radical route involving homolytic cleavage of the Cbenzyl–O bond to give a triplet ion–neutral complex, followed by decarboxylative coupling.
Ion mobility spectrometry provides information about molecular structures of ions. Hence, high resolving power allows separation of isomers which is of major interest in several applications. In this work, we couple our high-resolution ion mobility spectrometer (IMS) with a resolving power of Rp?=?100 to a time-of-flight mass spectrometer (TOF-MS). Besides, the benefit of an increased resolving power such an IMS-MS also helps analyzing and understanding the ionization processes in IMS. Usually, the coupling between IMS and TOF-MS is realized by synchronizing data acquisition of the IMS and MS resulting in two-dimensional data containing ion mobility and mass spectra. However, due to peak widths of less than 100 μs in our high-resolution IMS, this technique is not practicable due to significant peak broadening during the ion transfer into the MS and an insufficient data acquisition rate of the MS. Thus, a novel but simple interface between the IMS and MS has been designed which minimizes ion losses, allows recording of ion mobility at full IMS resolving power, and enables a shuttered transmission of ions into the MS. The interface is realized by replacing the Faraday plate used in IMS by a Faraday grid that is shielded by two additional aperture grids. For demonstration, positive product ions of benzene, toluene, and m-xylene in air are investigated. The IMS is equipped with a radioactive 3H source. Besides the well-known product ions M+ and M·NO+, a dimer ion is also observed for benzene and toluene, consisting of two molecules and three further hydrogen atoms.
Systematic manipulation of ionic-outcome in laser-cluster interaction process has been realized for studies carried out on tetramethyltin (TMT) clusters under picosecond laser conditions, determined by choice of laser wavelength and intensity. As a function of laser intensity, TMT clusters exhibit gradual enhancement in overall ionization of its cluster constituents, up to a saturation level of ionization, which was distinct for different wavelengths (266, 355, and 532 nm). Simultaneously, systematic appearance of higher multiply charged atomic ions and shift in relative abundance of multiply charged atomic ions towards higher charge state was observed, using time-of-flight mass spectrometer. At saturation level, multiply charged atomic ions up to (C2+, Sn2+) at 266 nm, (C4+, Sn4+) at 355 nm, and (C4+, Sn6+) at 532 nm were detected. In addition, at 355 nm intra-cluster ion chemistry within the ionized cluster leads to generation of molecular hydrogen ion (H2+) and triatomic molecular hydrogen ion (H3+). Generation of multiply charged atomic ions is ascribed to efficient coupling of laser pulse with the cluster media, facilitated by inner-ionized electrons produced within the cluster, at the leading edge of laser pulse. Role of inner-ionized electrons is authenticated by measuring kinetic energy distribution of electrons liberated upon disintegration of excessively ionized cluster, under the influence of picosecond laser pulse.
Electron capture dissociation (ECD) and electron transfer dissociation (ETD) in metal-peptide complexes are dependent on the metal cation in the complex. The divalent transition metals Ni2+, Cu2+, and Zn2+ were used as charge carriers to produce metal-polyhistidine complexes in the absence of remote protons, since these metal cations strongly bind to neutral histidine residues in peptides. In the case of the ECD and ETD of Cu2+-polyhistidine complexes, the metal cation in the complex was reduced and the recombination energy was redistributed throughout the peptide to lead a zwitterionic peptide form having a protonated histidine residue and a deprotonated amide nitrogen. The zwitterion then underwent peptide bond cleavage, producing a and b fragment ions. In contrast, ECD and ETD induced different fragmentation processes in Zn2+-polyhistidine complexes. Although the N–Cα bond in the Zn2+-polyhistidine complex was cleaved by ETD, ECD of Zn2+-polyhistidine induced peptide bond cleavage accompanied with hydrogen atom release. The different fragmentation modes by ECD and ETD originated from the different electronic states of the charge-reduced complexes resulting from these processes. The details of the fragmentation processes were investigated by density functional theory.
In a study of the metal-related ion generation mechanism in matrix-assisted laser desorption ionization (MALDI), crystals of matrix used in MALDI were grown from matrix- and salt-containing solutions. The intensities of metal ion and metal adducts of the matrix ion obtained from unwashed crystals were higher than those from crystals washed with deionized water, indicating that metal ions and metal adducts of the matrix ions are mainly generated from the surface of crystals. The contributions of preformed metal ions and metal adducts of the matrix ions inside the matrix crystals were minor. Metal adducts of the matrix and analyte ion intensities generated from a mixture of dried matrix, salt, and analyte powders were similar to or higher than those generated from the powder of dried droplet crystals, indicating that the contributions of the preformed metal adducts of the matrix and analyte ions were insignificant. Correlation between metal-related ion intensity fluctuation and protonated ion intensity fluctuation was observed, indicating that the generation mechanism of the metal-related ions is similar to that of the protonated ions. Because the thermally induced proton transfer model effectively describes the generation of the protonated ions, we suggest that metal-related ions are mainly generated from the salt dissolution in the matrix melted by the laser.
We investigate the tandem mass spectrometry of regiospecifically labeled, deprotonated sucrose analytes. We utilize density functional theory calculations to model the pertinent gas-phase fragmentation chemistry of the prevalent glycosidic bond cleavages (B1-Y1 and C1-Z1 reactions) and compare these predictions to infrared spectroscopy experiments on the resulting B1 and C1 product anions. For the C1 anions, barriers to interconversion of the pyranose [α-glucose-H]?, C1 anions to entropically favorable ring-open aldehyde-terminated forms were modest (41 kJ mol?1) consistent with the observation of a band assigned to a carbonyl stretch at ~?1680–1720 cm?1. For the B1 anions, our transition structure calculations predict the presence of both deprotonated 1,6-anhydroglucose and carbon 2-ketone ((4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)dihydro-2H-pyran-3(4H)-one) anion structures, with the latter predominating. This hypothesis is supported by our spectroscopic data which show diagnostic bands at 1600, 1674, and 1699 cm?1 (deprotonated carbon 2-ketone structures), and at ~?1541 cm?1 (both types of structure) and RRKM rate calculations. The deprotonated carbon 2-ketone structures are also the lowest energy product B1 anions.
A method for continuously monitoring the performance of electrospray ionization without the addition of hardware or chemistry to the system is demonstrated. In the method, which we refer to as SprayDx, cluster ions with solvent vapor natively formed by electrospray are followed throughout the collection of liquid chromatography–selected reaction monitoring data. The cluster ion extracted ion chromatograms report on the consistency of the ion formation and detection system. The data collected by the SprayDx method resemble the data collected for postcolumn infusion of analyte. The response of the cluster ions monitored reports on changes in the physical parameters of the ion source such as voltage and gas flow. SprayDx is also observed to report on ion suppression in a fashion very similar to a postcolumn infusion of analyte. We anticipate the method finding utility as a continuous readout on the performance of electrospray and other atmospheric pressure ionization processes.
We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states.
Ion mobility measurements of product ions were used to characterize the collisional cross section (CCS) of various complex lipid [M-H]? ions using traveling wave ion mobility mass spectrometry (TWIMS). TWIMS analysis of various product ions derived after collisional activation of mono- and dihydroxy arachidonate metabolites was found to be more complex than the analysis of intact molecular ions and provided some insight into molecular mechanisms involved in product ion formation. The CCS observed for the molecular ion [M-H]? and certain product ions were consistent with a folded ion structure, the latter predicted by the proposed mechanisms of product ion formation. Unexpectedly, product ions from [M-H-H2O-CO2]? and [M-H-H2O]? displayed complex ion mobility profiles suggesting multiple mechanisms of ion formation. The [M-H-H2O]? ion from LTB4 was studied in more detail using both nitrogen and helium as the drift gas in the ion mobility cell. One population of [M-H-H2O]? product ions from LTB4 was consistent with formation of covalent ring structures, while the ions displaying a higher CCS were consistent with a more open-chain structure. Using molecular dynamics and theoretical CCS calculations, energy minimized structures of those product ions with the open-chain structures were found to have a higher CCS than a folded molecular ion structure. The measurement of product ion mobility can be an additional and unique signature of eicosanoids measured by LC-MS/MS techniques.
In this work, we show the advantages of label-free, tridimensional mass spectrometry imaging using dual beam analysis (25 keV Bi3+) and depth profiling (20 keV with a distribution centered at Ar1500+) coupled to time of flight secondary ion mass spectrometry (3D-MSI-TOF-SIMS) for the study of A-172 human glioblastoma cell line treated with B-cell lymphoma 2 (Bcl-2) inhibitor ABT-737. The high spatial (~250 nm) and high mass resolution (m/Δm ~10,000) of TOF-SIMS permitted the localization and identification of the intact, unlabeled drug molecular ion (m/z 811.26 C42H44ClN6O5S2– [M – H]–) as well as characteristic fragment ions. We propose a novel approach based on the inspection of the drug secondary ion yield, which showed a good correlation with the drug concentration during cell treatment at therapeutic dosages (0–200 μM with 4 h incubation). Chemical maps using endogenous molecular markers showed that the ABT-737 is mainly localized in subsurface regions and absent in the nucleus. A semiquantitative workflow is proposed to account for the biological cell diversity based on the spatial distribution of endogenous molecular markers (e.g., nuclei and cytoplasm) and secondary ion confirmation based on the ratio of drug-specific fragments to molecular ion as a function of the therapeutic dosage.
Differential ion mobility spectrometry (DMS) spatially separates ions in the gas phase using the mobility differences of the ions under applied low and high electric fields. The use of DMS as an ion filter (or ion selector) prior to mass spectrometry analysis has been compromised by the limited ion transmission efficiency. This paper reports enhancement of the DMS-MS sensitivity and signal stability using a modified CaptiveSpray? source. In terms of the ion sampling and transmission efficiency, the modified CaptiveSpray source swept ~ 89% of the ions generated by the tapered capillary through the DMS device (compared to ~ 10% with a conventional microspray source). The signal fluctuation improved from 11.7% (relative standard deviation, RSD) with microspray DMS-MS to 3.6% using CaptiveSpray-DMS-MS. Coupling of LC to DMS-MS via the modified CaptiveSpray source was simple and robust. Using DMS as a noise-filtering device, LC-DMS-MS performed better than conventional LC-MS for analyzing a BSA digest standard. Although LC-DMS-MS had a lower sequence coverage (55%), a higher Mascot score (283) was obtained compared to those of LC-MS (sequence coverage 65%; Mascot score 192) under the same elution conditions. The improvement in the confidence of the search result was attributed to the preferential elimination of noise ions.
Extracted arrival time distributions of negative ion CID-derived fragments produced prior to traveling-wave ion mobility separation were evaluated for their ability to provide structural information on N-linked glycans. Fragmentation of high-mannose glycans released from several glycoproteins, including those from viral sources, provided over 50 fragments, many of which gave unique collisional cross-sections and provided additional information used to assign structural isomers. For example, cross-ring fragments arising from cleavage of the reducing terminal GlcNAc residue on Man8GlcNAc2 isomers have unique collision cross-sections enabling isomers to be differentiated in mixtures. Specific fragment collision cross-sections enabled identification of glycans, the antennae of which terminated in the antigenic α-galactose residue, and ions defining the composition of the 6-antenna of several of the glycans were also found to have different cross-sections from isomeric ions produced in the same spectra. Potential mechanisms for the formation of the various ions are discussed and the estimated collisional cross-sections are tabulated.
Miniaturised field asymmetric waveform ion mobility spectrometry (FAIMS), combined with mass spectrometry (MS), has been applied to the study of self-assembling, noncovalent supramolecular complexes of 3-methylxanthine (3-MX) in the gas phase. 3-MX forms stable tetrameric complexes around an alkali metal (Na+, K+) or ammonium cation, to generate a diverse array of complexes with single and multiple charge states. Complexes of (3-MX)n observed include: singly charged complexes where n = 1–8 and 12 and doubly charged complexes where n = 12–24. The most intense ions are those associated with multiples of tetrameric units, where n = 4, 8, 12, 16, 20, 24. The effect of dispersion field on the ion intensities of the self-assembled complexes indicates some fragmentation of higher order complexes within the FAIMS electrodes (in-FAIMS dissociation), as well as in-source collision induced dissociation within the mass spectrometer. FAIMS-MS enables charge state separation of supramolecular complexes of 3-MX and is shown to be capable of separating species with overlapping mass-to-charge ratios. FAIMS selected transmission also results in an improvement in signal-to-noise ratio for low intensity complexes and enables the visualization of species undetectable without FAIMS.
A detailed energy-resolved study of the fragmentation reactions of protonated histidine-containing peptides and their b2 ions has been undertaken. Density functional theory calculations were utilized to predict how the fragmentation reactions occur so that we might discern why the mass spectra demonstrated particular energy dependencies. We compare our results to the current literature and to synthetic b2 ion standards. We show that the position of the His residue does affect the identity of the subsequent b2 ion (diketopiperazine versus oxazolone versus lactam) and that energy-resolved CID can distinguish these isomeric products based on their fragmentation energetics. The histidine side chain facilitates every major transformation except trans-cis isomerization of the first amide bond, a necessary prerequisite to diketopiperazine b2 ion formation. Despite this lack of catalyzation, trans-cis isomerization is predicted to be facile. Concomitantly, the subsequent amide bond cleavage reaction is rate-limiting.
We report on the performance of a cryogenic 2D linear ion trap (cryoLIT) that is shown to be mass-selective in the temperature range of 17–295 K. As the cryoLIT is cooled, the ejection voltages during the mass instability scan decrease, which results in an effective mass shift to lower m/z relative to room temperature. This is attributed to a decrease in trap radius caused by thermal contraction. Additionally, the cryoLIT generates reproducible mass spectra from day-to-day, and is capable of performing stored waveform inverse Fourier transform (SWIFT) mass isolation of fragile N2-tagged ions for the purpose of background-free infrared dissociation spectroscopy.
