Vitamin D compounds are secosteroids, which are best known for their role in bone health. More recent studies have shown that vitamin D metabolites and catabolites such as dihydroxylated species (e.g., 1,25- and 24,25-dihydroxyvitamin D3) play key roles in the pathologies of various diseases. Identification of these isomers by mass spectrometry is challenging and currently relies on liquid chromatography, as the isomers exhibit virtually identical product ion spectra under collision induced dissociation conditions. Here, we developed a simple MALDI-CID method that utilizes ion activation of reactive analyte/matrix adducts to distinguish isomeric dihydroxyvitamin D3 species, without the need for chromatography separation or chemical derivatization techniques. Specifically, reactive 1,5-diaminonaphthalene adducts of dihydroxyvitamin D3 compounds formed during MADI were activated and specific cleavages in the secosteroid’s backbone structure were achieved that produced isomer-diagnostic fragment ions.
Corona discharge ionization sources are often used in ion mobility spectrometers (IMS) when a non-radioactive ion source with high ion currents is required. Typically, the corona discharge is followed by a reaction region where analyte ions are formed from the reactant ions. In this work, we present a simple yet sufficiently accurate model for predicting the ion current available at the end of this reaction region when operating at reduced pressure as in High Kinetic Energy Ion Mobility Spectrometers (HiKE-IMS) or most IMS-MS instruments. It yields excellent qualitative agreement with measurement results and is even able to calculate the ion current within an error of 15%. Additional interesting findings of this model are the ion current at the end of the reaction region being independent from the ion current generated by the corona discharge and the ion current in High Kinetic Energy Ion Mobility Spectrometers (HiKE-IMS) growing quadratically when scaling down the length of the reaction region.
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
The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-μm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution.
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.
Dynamic reactive ionization (DRI) utilizes a reactive molecule, HCl, which is doped into an Ar cluster projectile and activated to produce protons at the bombardment site on the cold sample surface with the presence of water. The methodology has been shown to enhance the ionization of protonated molecular ions and to reduce salt suppression in complex biomatrices. In this study, we further examine the possibility of obtaining improved quantitation with DRI during depth profiling of thin films. Using a trehalose film as a model system, we are able to define optimal DRI conditions for depth profiling. Next, the strategy is applied to a multilayer system consisting of the polymer antioxidants Irganox 1098 and 1010. These binary mixtures have demonstrated large matrix effects, making quantitative SIMS measurement not feasible. Systematic comparisons of depth profiling of this multilayer film between directly using GCIB, and under DRI conditions, show that the latter enhances protonated ions for both components by 4- to ~15-fold, resulting in uniform depth profiling in positive ion mode and almost no matrix effect in negative ion mode. The methodology offers a new strategy to tackle the matrix effect and should lead to improved quantitative measurement using SIMS.
Gas phase infrared dissociation spectra of the radical cation, deprotonated and protonated forms of the hormone melatonin, and its complexes with alkali (Li+, Na+, and K+) and alkaline earth metal ions (Mg2+, Ca2+, and Sr2+) are measured in the spectral range 800–1800 cm?1. Minimum energy geometries calculated at the B3LYP/LACVP++** level are used to assign structural motifs to absorption bands in the experimental spectra. The melatonin anion is deprotonated at the indole-N. The indole-C linking the amide chain is the most favored protonation site. Comparisons between the experimental and calculated spectra for alkali and alkaline earth metal ion complexes reveal that the metal ions interact similarly with the amide and methoxy oxygen atoms. The amide I band undergoes a red shift with increasing charge density of the metal ion and the amide II band shows a concomitant blue shift. Another binding motif in which the metal ions interact with the amide-O and the π-electron cloud of the aromatic group is identified but is higher in energy by at least 18 kJ/mol. Melatonin is deprotonated at the amide-N with Mg2+ and the metal ion coordinates to the amide-N and an indole-C or the methoxy-O. These results provide information about the intrinsic binding of metal ions to melatonin and combined with future studies on solvated melatonin-metal ion complexes may help elucidate the solvent effects on metal ion binding in solution and the biochemistry of melatonin. These results also serve as benchmarks for future theoretical studies on melatonin-metal ion interactions.
The acyl substitution reactions between 1-hydroxy-7-aza-benzotriazole (HOAt)/1-hydroxy-benzotriazole (HOBt) ester reagents and nucleophilic side chains on peptides have been demonstrated in the gas phase via ion/ion reactions. The HOAt/HOBt ester reagents were synthesized in solution and ionized via negative nano-electrospray ionization. The anionic reagents were then reacted with doubly protonated model peptides containing amines, guanidines, and imidazoles in the gas phase. The complexes formed in the reaction cell were further probed with ion trap collision induced dissociation (CID) yielding either a covalently modified analyte ion or a proton transfer product ion. The covalent reaction yield of HOAt/HOBt ester reagents was demonstrated to be higher than the yield with N-hydroxysuccinimide (NHS) ester reagents over a range of equivalent conditions. Density functional theory (DFT) calculations were performed with a primary amine model system for both triazole-ester and NHS-ester reactants, which indicated a lower transition state barrier for the former reagent, consistent with experiments. The work herein demonstrates that the triazole-ester reagents are more reactive, and therefore less selective, than the analogous NHS-ester reagent. As a consequence, the triazole-ester reagents are the first to show efficient reactivity with unprotonated histidine residues in the gas phase. For all nucleophilic sites and all reagents, covalent reactions are favored under long time, low amplitude activation conditions. This work presents a novel class of reagents capable of gas-phase conjugation to nucleophilic sites in analyte ions via ion/ion chemistry.
A novel method for the simultaneous detection of ingredients in pharmaceutical applications such as creams and lotions was developed. An ultrasonic atomizer has been used to produce a mist containing ingredients. The analyte molecules in the mist can be ionized by using direct analysis in real time (DART) at lower temperature than traditionally used, and we thus solved the problem of normal DART-MS measurement using a high-temperature gas. Thereby, molecular-related ions of heat-unstable components and nonvolatile components became detectable. The deprotonated molecular ion of glycyrrhizic acid (m/z 821), which is unstable at high temperatures, was detected without pyrolysis by ultrasonic mist–DART-MS using unheated helium gas, although it was not detected by normal DART-MS using heated helium gas. The cationized molecular ions of derivatives of polyethylene glycol fatty acid monoesters, which are nonvolatile compounds, were also detected as m/z peaks observed from 800 to 2300. Although the protonated molecular ion of tocopherol acetate was not detected in ionization by ultrasonic mist, it was detected by ultrasonic mist–DART-MS even in the emulsion. It was not necessary to dissolve a sample completely to detect its ions. This method enabled us to obtain the composition of pharmaceutical applications simply and rapidly.
The utility of sodium ion adducts produced by matrix-assisted laser desorption ionization for the quantification of analytes with multiple oxygen atoms was evaluated. Uses of homogeneous solid samples and temperature control allowed the acquisition of reproducible spectra. The method resulted in a direct proportionality between the ion abundance ratio I([A?+?Na]+)/I([M?+?Na]+) and the analyte concentration, which could be used as a calibration curve. This was demonstrated for carbohydrates, glycans, and polyether diols with dynamic range exceeding three orders of magnitude.
The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer’s disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers.
Ionization processes have been discovered by which small and large as well as volatile and nonvolatile compounds are converted to gas-phase ions when associated with a matrix and exposed to sub-atmospheric pressure. Here, we discuss experiments further defining these simple and unexpected processes. Charge separation is found to be a common process for small molecule chemicals, solids and liquids, passed through an inlet tube from a higher to a lower pressure region, with and without heat applied. This charge separation process produces positively- and negatively-charged particles with widely different efficiencies depending on the compound and its physical state. Circumstantial evidence is presented suggesting that in the new ionization process, charged particles carry analyte into the gas phase, and desolvation of these particles produce the bare ions similar to electrospray ionization, except that solid particles appear likely to be involved. This mechanistic proposition is in agreement with previous theoretical work related to ion emission from ice.
Immonium ions are commonly observed in the high energy fragmentation of peptide ions. In a MALDI-TOF/TOF mass spectrometer, singly charged peptides photofragmented with 157 nm VUV light yield a copious abundance of immonium ions, especially those from aromatic residues. However, their intensities may vary from one peptide to another. In this work, the effect of varying amino acid position, peptide length, and peptide composition on immonium ion yield is investigated. Internal immonium ions are found to have the strongest intensity, whereas immonium ions arising from C-terminal residues are the weakest. Peptide length and competition among residues also strongly influence the immonium ion production. Quantum calculations provide insights about immonium ion structures and the fragment ion conformations that promote or inhibit immonium ion formation.
The utility of novel functionalized nanodiamonds (NDs) as matrices for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) is described herein. MALDI-MS analysis of small organic compounds (<1000 Da) is typically complex because of interferences from numerous cluster ions formed when using conventional matrices. To expand the use of MALDI for the analysis of small molecules, novel matrices were designed by covalently linking conventional matrices (or a lysine moiety) to detonated NDs. Four new functionalized NDs were evaluated for their ionization capabilities using five pharmaceuticals with varying molecular structures. Two ND matrices were able to ionize all tested pharmaceuticals in the negative ion mode, producing the deprotonated ions [M – H]–. Ion intensity for target analytes was generally strong with enhanced signal-to-noise ratios compared with conventional matrices. The negative ion mode is of great importance for biological samples as interference from endogenous compounds is inherently minimized in the negative ion mode. Since the molecular structures of the tested pharmaceuticals did not suggest that negative ion mode would be preferable, this result magnifies the importance of these findings. On the other hand, conventional matrices primarily facilitated the ionization as expected in the positive ion mode, producing either the protonated molecules [M + H]+ or cationic adducts (typically producing complex spectra with numerous adduct peaks). The data presented in this study suggests that these matrices may offer advantages for the analysis of low molecular weight pharmaceuticals/metabolites.
Elucidation of ion dissociation patterns is particularly important to structural analysis by mass spectrometry (MS). However, typically, only the charged fragments from an ion dissociation event are detected in tandem MS experiments; neutrals are not identified. In recent years, we have developed an atmospheric pressure thermal dissociation (APTD) technique that can be applied to dissociate ions at atmosphere pressure and thus provide one way to characterize neutral fragments. In this paper, we focus on the detection of neutral CO resulting from amino acid and peptide ion dissociation. In the first set of experiments, several protonated amino acids (e.g., +?1 ion of phenylalanine) were found to undergo loss of a neutral (s) of total mass 46 Da, a process leading to iminium ion formation. We successfully detected the neutral species CO by using a CO sensor, UV-Vis and MS analysis following selective CO trapping with a rhodium complex. The capture of CO from dissociation of protonated amino acids supports the assignment of the loss of 46 Da to neutral losses of CO and H2O, rather than loss of formaldehyde or dihydroxycarbene, other possible fragmentation pathways that have been subject of debate for a long time. In a second experiment, we used the APTD method in combination with the CO detection technique, to demonstrate the formation of CO in the conversion of b ions to a ions during peptide ion dissociations. These results showed the potential of APTD in the elucidation of ion dissociation mechanisms, using simple home-built apparatus.
A potassium-containing hexaazamacrocyclic dication, [M?H?K]2+, is able to add in the gas phase mono- and dicarboxylate anions as well as inorganic anions by forming the corresponding monocharged adducts, the structure of which markedly depends on the basicity of the anion. With anions, such as acetate or fluoride, the neutral hexaazamacrocycle M acts as an acceptor of monosolvated K+ ion. With less basic anions, such as trifluoroacetate or chloride, the protonated hexaazamacrocycle [M?H]+ performs the unusual functions of an acceptor of contact K+/anion pairs.
Paper spray mass spectrometry is a method for the direct analysis of biofluid samples in which extraction of analytes from dried biofluid spots and electrospray ionization occur from the paper on which the dried sample is stored. We examined matrix effects in the analysis of small molecule drugs from urine, plasma, and whole blood. The general method was to spike stable isotope labeled analogs of each analyte into the spray solvent, while the analyte itself was in the dried biofluid. Intensity of the labeled analog is proportional to ionization efficiency, whereas the ratio of the analyte intensity to the labeled analog in the spray solvent is proportional to recovery. Ion suppression and recovery were found to be compound- and matrix-dependent. Highest levels of ion suppression were obtained for poor ionizers (e.g., analytes lacking basic aliphatic amine groups) in urine and approached –90%. Ion suppression was much lower or even absent for good ionizers (analytes with aliphatic amines) in dried blood spots. Recovery was generally highest in urine and lowest in blood. We also examined the effect of two experimental parameters on ion suppression and recovery: the spray solvent and the sample position (how far away from the paper tip the dried sample was spotted). Finally, the change in ion suppression and analyte elution as a function of time was examined by carrying out a paper spray analysis of dried plasma spots for 5 min by continually replenishing the spray solvent.
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.
