Gas-phase hydrogen deuterium exchange (HDX), collision cross section (CCS) measurement, and molecular dynamics simulation (MDS) techniques were utilized to develop and compare three methods for estimating the relative surface area exposure of separate peptide chains within bovine insulin ions. Electrosprayed [M – 3H]3? and [M – 5H]5? insulin ions produced a single conformer type with respective collision cross sections of 528 ± 5 Å2 and 808 ± 2 Å2. [M – 4H]4? ions were comprised of more compact (Ω = 676 ± 3 Å2) and diffuse (i.e., more elongated, Ω = 779 ± 3 Å2) ion conformer types. Ions were subjected to HDX in the drift tube using D2O as the reagent gas. Collision-induced dissociation was used to fragment mobility-selected, isotopically labeled [M – 4H]4? and [M – 5H]5? ions into the protein subchains. Deuterium uptake levels of each chain can be explained by limited inter-chain isotopic scrambling upon collisional activation. Using nominal ion structures from MDS and a hydrogen accessibility model, the deuterium uptake for each chain was correlated to its exposed surface area. In separate experiments, the per-residue deuterium content for the protonated and deprotonated ions of the synthetic peptide KKDDDDDIIKIIK were compared. The differences in deuterium content indicated the regional HDX accessibility for cations versus anions. Using ions of similar conformational type, this comparison highlights the complementary nature of HDX data obtained from positive- and negative-ion analysis.
The dominant gas-phase conformer of [M+3H]3+ ions of the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK has been examined with ion mobility spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass spectrometry (MS) techniques. The [M+3H]3+ peptide ions are observed predominantly as a relatively compact conformer type. Upon subjecting these ions to electron transfer dissociation (ETD), the level of protection for each amino acid residue in the peptide sequence is assessed. The overall per-residue deuterium uptake is observed to be relatively more efficient for the neutral residues than for the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. In comparison, the N-terminal and C-terminal regions of the serine peptide show greater relative protection compared with interior residues. Molecular dynamics (MD) simulations have been used to generate candidate structures for collision cross section and HDX reactivity matching. Hydrogen accessibility scoring (HAS) for select structural candidates from MD simulations has been used to suggest conformer types that could contribute to the observed HDX patterns. The results are discussed with respect to recent studies employing extensive MD simulations of gas-phase structure establishment of a peptide system.
To date, only a limited number of reports are available on structural variants of multiply-charged b-fragment ions. We report on observed bimodal gas-phase hydrogen/deuterium exchange (HDX) reaction kinetics and patterns for substance P b102+ that point to presence of isomeric structures. We also compare HDX reactions, post-ion mobility/collision-induced dissociation (post-IM/CID), and sustained off-resonance irradiation-collision induced dissociation (SORI-CID) of substance P b102+ and a cyclic peptide with an identical amino acid (AA) sequence order to substance P b10. The observed HDX patterns and reaction kinetics and SORI-CID pattern for the doubly charged head-to-tail cyclized peptide were different from either of the presumed isomers of substance P b102+, suggesting that b102+ may not exist exclusively as a head-to-tail cyclized structure. Ultra-high mass measurement accuracy was used to assign identities of the observed SORI-CID fragment ions of substance P b102+; over 30 % of the observed SORI-CID fragment ions from substance P b102+ had rearranged (scrambled) AA sequences. Moreover, post-IM/CID experiments revealed the presence of two conformer types for substance P b102+, whereas only one conformer type was observed for the head-to-tail cyclized peptide. We also show that AA sequence scrambling from CID of doubly-charged b-fragment ions is not unique to substance P b102+.
Stereochemistry plays an important role in biochemistry, particularly in therapeutic applications. Indeed, enantiomers have different biological activities, which can have important consequences. Many analytical techniques have been developed in order to allow the identification and the separation of stereoisomers. Here, we focused our work on the study of small diastereomers using the coupling of traveling wave ion mobility and mass spectrometry (TWIMS-MS) as a new alternative for stereochemistry study. In order to optimize the separation, the formation of adducts between diastereomers (M) and different alkali cations (X) was carried out. Thus, monomers [M + X]+ and multimers [2M + X]+ and [3M + X]+ ions have been studied from both experimental and theoretical viewpoints. Moreover, it has been shown that the study of the multimer [2Y + M + Li]+ ion, in which Y is an auxiliary diastereomeric ligand, allows the diastereomers separation. The combination of cationization, multimers ions formation, and IM-MS is a novel and powerful approach for the diastereomers identification. Thus, by this technique, diastereomers can be identified although they present very close conformations in gaseous phase. This work presents the first TWIMS-MS separation of diastereomers, which present very close collision cross section thanks to the formation of multimers and the use of an auxiliary diastereomeric ligand.
The solution dependence of gas-phase unfolding for ubiquitin [M + 7H]7+ ions has been studied by ion mobility spectrometry-mass spectrometry (IMS-MS). Different acidic water:methanol solutions are used to favor the native (N), more helical (A), or unfolded (U) solution states of ubiquitin. Unfolding of gas-phase ubiquitin ions is achieved by collisional heating and newly formed structures are examined by IMS. With an activation voltage of 100 V, a selected distribution of compact structures unfolds, forming three resolvable elongated states (E1-E3). The relative populations of these elongated structures depend strongly on the solution composition. Activation of compact ions from aqueous solutions known to favor N-state ubiquitin produces mostly the E1 type elongated state, whereas activation of compact ions from methanol containing solutions that populate A-state ubiquitin favors the E3 elongated state. Presumably, this difference arises because of differences in precursor ion structures emerging from solution. Thus, it appears that information about solution populations can be retained after ionization, selection, and activation to produce the elongated states. These data as well as others are discussed. Figure
A novel quartz crystal microbalance (QCM) sensor has been developed for highly selective and sensitive detection of Pb2+ by exploiting the catalytic effect of Pb2+ ions on the leaching of gold nanoparticles from the surface of a QCM sensor. The use of self-assembled gold nanoparticles (AuNPs) strongly enlarges the size of the interface and thus amplifies the analytical response resulting from the loss of mass. This results in a very low detection limit for Pb2+ (30 nM). The high selectivity is demonstrated by studying the effect of potentially interfering ions both in the absence and presence of Pb2+ ions. This simple and well reproducible sensor was applied to the determination of lead in the spiked drinking water. This work provides a novel strategy for fabricating QCM sensors towards Pb2+ in real samples. Figure
Negative-ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra and tandem mass spectra of flavonoid mono-O-glycosides showed the irregular signals that were 1 and/or 2 Da smaller than the parent deprotonated molecules ([M – H]–) and the sugar-unit lost fragment ions ([M – Sugar – H]–). The 1 and/or 2 Da mass shifts are generated with the removing of a neutral hydrogen radical (H*), and/or with the homolytic cleavage of the glycosidic bond, such as [M – H* – H]–, [M – Sugar – H* – H]–, and [M – Sugar – 2H* – H]–. It was revealed that the hydrogen radical removes from the phenolic hydroxy groups on the flavonoids, not from the sugar moiety, because the flavonoid backbones themselves absorb the laser. The glycosyl positions depend on the extent of the hydrogen radical removals and that of the homolytic cleavage of the glycosidic bonds. Flavonoid mono-glycoside isomers were distinguished according to their TOF MS and tandem mass spectra.
The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M?+?H?+?O]+), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M?+?H?+?O]+ product is observed at a much greater abundance than the proton transfer product (viz., [M?+?H]+). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to ‘label’ methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.
A kiloelectronvolt beam of helium ions is used to ionize and fragment precursor peptide ions starting in the 1+ charge state. The electron affinity of helium cations (24.6 eV) exceeds the ionization potential of protonated peptides and can therefore be used to abstract an electron from—or charge exchange with—the isolated precursor ions. Kiloelectronvolt energies are used, (1) to overcome the Coulombic repulsion barrier between the cationic reactants, (2) to overcome ion-defocussing effects in the ion trap, and (3) to provide additional activation energy. Charge transfer dissociation (CTD) of the [M+H]+ precursor of Substance P gives product ions such as [M+H]2+? and a dominant series of a ions in both the 1+ and 2+ charge states. These observations, along with the less-abundant a + 1 ions, are consistent with ultraviolet photodissociation (UVPD) results of others and indicate that C–Cα cleavages are possible through charge exchange with helium ions. Although the efficiencies and timescale of CTD are not yet suitable for on-line chromatography, this new approach to ion activation provides an additional potential tool for the interrogation of gas phase ions. Graphical Abstract
Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein–protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods. Figure
A new method for measuring the ion velocity distribution using an internal matrix-assisted laser desorption/ionization (MALDI) source Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer is described. The method provides the possibility of studying ion velocities without any influence of electric fields in the direction of the instrument axis until the ions reach the ICR cell. It also allows to simultaneously account for and to estimate not only the velocity distribution but the angular distribution as well. The method was demonstrated using several types of compounds in laser desorption/ionization (LDI) mode. Graphical Abstract
During their travel inside a traveling wave ion mobility cell (TW IMS), ions are susceptible to heating because of the presence of high intensity electric fields. Here, we report effective temperatures Teff,vib obtained at the injection and inside the mobility cell of a SYNAPT G2 HDMS spectrometer for different probe ions: benzylpyridinium ions and leucine enkephalin. Using standard parameter sets, we obtained a temperature of ~800 K at injection and 728?±?2 K into the IMS cell for p-methoxybenzylpyridinium. We found that Teff,vib inside the cell was dependent on the separation parameters and on the nature of the analyte. While the mean energy of the Boltzmann distributions increases with ion size, the corresponding temperature decreases because of increasing numbers of vibrational normal modes. We also investigated conformational rearrangements of 7+ ions of cytochrome c and reveal isomerization of the most compact structure, therefore highlighting the effects of weak heating on the gas-phase structure of biologically relevant ions.
In this report, a method for in-source hydrogen/deuterium (H/D) exchange at atmospheric pressure is reported. The method was named atmospheric pressure photo ionization hydrogen/deuterium exchange mass spectrometry (APPI HDX MS). H/D exchange was performed by mixing samples dissolved in toluene with CH3OD solvent and analyzing the mixture using atmospheric pressure photo ionization mass spectrometry (APPI-MS). The APPI HDX spectra obtained with contact times between the analyte solution and methanol-OD (CH3OD) of?<?0.5 s or 1 h showed the same pattern of H/D exchange. Therefore, it was concluded that APPI HDX occurred in the source but not in the solution. The proposed method does not require a specific type of mass spectrometer and can be performed at atmospheric pressure. H/D exchange can be performed in any laboratory with a mass spectrometer and a commercial APPI source. Using this method, multiple H/D exchanges of aromatic hydrogen and/or H/D exchange of active hydrogen were observed. These results demonstrated that H/D exchange can be used to distinguish between isomers containing primary, secondary, and tertiary amines, as well as pyridine and pyrrole functional groups.
Simulations show that significant ion losses occur within the commercial electrospray ionization-field asymmetric waveform ion mobility spectrometer (ESI-FAIMS) interface owing to an angular desolvation gas flow and because of the impact of the FAIMS carrier gas onto the inner rf (radio frequency) electrode. The angular desolvation gas flow diverts ions away from the entrance plate orifice while the carrier gas annihilates ions onto the inner rf electrode. A novel ESI-FAIMS interface is described that optimizes FAIMS gas flows resulting in large improvements in transmission. Simulations with the bromochloroacetate anion showed an improvement of ~9-fold to give ~70% overall transmission). Comparable transmission improvements were attained experimentally for six peptides (2+) in the range of m/z 404.2 to 653.4 at a chromatographic flow rate of 300 nL/min. Selected ion chromatograms (SIC) from nano-LC-FAIMS-MS analyses showed 71% (HLVDEPQNLIK, m/z 653.4, 2+) to 95% (LVNELTEFAK, m/z 582.3, 2+) of ion signal compared with ion signal in the SIC from LC-MS analysis. IGSEVYHNLK (580.3, 2+) showed 24% more ion signal compared with LC-MS and is explained by enhanced desolvation in FAIMS. A 3–10 times lower limits of quantitation (LOQ) (<15% RSD) was achieved for chemical noise limited peaks with FAIMS. Peaks limited by ion statistics showed subtle improvement in RSD and yielded comparable LOQ to that attained with nano-LC-MS (without FAIMS). These improvements were obtained using a reduced FAIMS separation gap (from 2.5 to 1.5 mm) that results in a shorter residence time (13.2 ms?±?3.9 ms) and enables the use of a helium free transport gas (100% nitrogen). Graphical Abstract
Protonated nitroarginine, [RNO2 + H]+, which contains the nitroguanidine ‘explosophore,’ undergoes homolytic N – N nitro-imine bond cleavage to expel NO2? and form a radical cation of arginine in high yield (100 % relative abundance) upon low-energy collision-induced dissociation (CID). Other ionization states of nitroarginine, including [RNO2 - H]–, and a fixed-charge derivative of nitroarginine do not expel NO2? (<1 %), but instead dissociate via heterolytic bond cleavage with abundant losses of small molecules (N2O and H2N2O2) from the nitroguanidine group. The effects of proton mobility on the CID reactions of nitroarginine containing peptides was investigated for peptide derivatives of leucine enkephalin, including XYGGFLRNO2, X = D, G, K, and R, by examining the different protonation states: [M – H]–; [M + H]+; and [M + 2H]2+. For [M + H]+ containing the less basic N-terminal residues (X = D, G) and all [M + 2H]2+, mobile proton fragmentation reactions that result in peptide sequence ions dominate. In contrast, for peptides containing the basic N-terminal residues (R and K), the CID spectra of both the [M – H]– and [M + H]+ are dominated by the losses of small even-electron neutrals from the nitroarginine side-chain. The fraction of nitroguanidine directed fragmentation of the nitroarginine side chain that results in bond homolysis to form [XYGGFLR]+? by expulsion of NO2? increases by more than 10 times as the protonation state changes from [M – H]– (<10 %) to [M + 2H]2+ (ca. 90 %) and by about four times as the acidity of the [M + H]+N-terminal residue increases from R (19.0 %) to D (76.5 %). These results indicate that protonated peptides containing nitroarginine can undergo non-canonical mobile proton triggered radical fragmentation.
Fast-atom bombardment (FAB) mass spectrometry was used to investigate the interaction of proton and alkali metal ions with dinucleotide analogs such as T-n-T (T = thymine moiety, n = polyether chain, e.g., triethylene, tetraethylene, pentaethylene, and hexaethylene ether 1–4), A-n-T (A = adenine unit 5–8), and T-n-OMe (9–12) in 3-nitrobenzyl alcohol matrix. The [M + H]+ ion is the most abundant ion for the A-n-T series, whereas in 1–4 and 9–12 the (TC2H4)+ ion is the most abundant. Formation of [M + H -C2H4O]+ ions, a characteristic fragmentation of crown ethers under electron ionization, is observed for compounds 1–12 and is more pronounced in 6 and 7. An abundant [M ? H]? ion is observed for all the compounds studied under negative ion FAB due to the presence of the (-CO-NH-CO-) group of thymine, an indication of existence of intramolecular H bonding. The FAB mass spectra of 1–12 with alkali metal ions (Li+, Na+, K+, Rb+, and Cs+) showed formation of abundant metal-coordinated ions ([M + Met]+ and [TC2H4 + Met]+). Compounds 3, 4, 6, 7, and 10–12 showed ions due to the substitution of the thymine moiety by a hydroxyl group ([M + Met ? 108]+, Met = metal ion). For compound 3 alone, substitution of two thymine groups ([M + Met - 216]+) was observed. Metastable ion studies were used to elucidate the structures of these potentially significant ions, and the ion formule were confirmed with high resolution measurements. Selectivity toward metal complexation with ligand size was seen in the T-n-T and A-n-T series and was even more pronounced in A-n-T series. These dinucleotide analogs fall in the following order of chelation of alkali metal ions, acyclic glymes < dinucleotide analogs (acyclic glymes substituted with nitrogen bases) < crown ethers, which places them in perspective as receptor models. 相似文献
U.S. food imports have been increasing steadily for decades, intensifying the need for a rapid and sensitive screening technique. A method has been developed that uses foam disks to sample the surface of incoming produce. This work provides complimentary information to the extensive amount of published pesticide fragmentation data collected using LCMS systems (Sack et al. Journal of Agricultural and Food Chemistry, 59, 6383–6411, 2011; Mol et al. Analytical and Bioanalytical Chemistry, 403, 2891–2908, 2012). The disks are directly analyzed using transmission-mode direct analysis in real time (DART) ambient pressure desorption ionization coupled to a high resolution accurate mass-mass spectrometer (HRAM-MS). In order to provide more certainty in the identification of the pesticides detected, a library of accurate mass fragments and isotopes of the protonated parent molecular ion (the [M+H]+) has been developed. The HRAM-MS is equipped with a quadrupole mass filter, providing the capability of “data-dependent” fragmentation, as opposed to “all -ion” fragmentation (where all of the ions enter a collision chamber and are fragmented at once). A temperature gradient for the DART helium stream and multiple collision energies were employed to detect and fragment 164 pesticides of varying chemical classes, sizes, and polarities. The accurate mass information of precursor ([M+H]+ ion) and fragment ions is essential in correctly identifying chemical contaminants on the surface of imported produce. Additionally, the inclusion of isotopes of the [M+H]+ in the database adds another metric to the confirmation process. The fragmentation data were collected using a Q-Exactive mass spectrometer and were added to a database used to process data collected with an Exactive mass spectrometer, an instrument that is more readily available for this screening application. The commodities investigated range from smooth-skinned produce such as apples to rougher surfaces like broccoli. The minimal sample preparation and absence of chromatography has shortened the analysis time to about 15 min per sample, and the simplicity and robustness of the technique make it ideal for rapid screening.
