Project description:Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach.
Project description:This is the first report of imaging mass spectrometry (MS) from multiply charged ions at vacuum. Laserspray ionization (LSI) was recently extended to applications at vacuum producing electrospray ionization-like multiply charged ions directly from surfaces using a commercial intermediate pressure matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) MS instrument. Here, we developed a strategy to image multiply charged peptide ions. This is achieved by the use of 2-nitrophloroglucinol as matrix for spray deposition onto the tissue section and implementation of "soft" acquisition conditions including lower laser power and ion accelerating voltages similar to electrospray ionization-like conditions. Sufficient ion abundance is generated by the vacuum LSI method to employ IMS separation in imaging multiply charged ions obtained on a commercial mass spectrometer ion source without physical instrument modifications using the laser in the commercially available reflection geometry alignment. IMS gas-phase separation reduces the complexity of the ion signal from the tissue, especially for multiply charged relative to abundant singly charged ions from tissue lipids. We show examples of LSI tissue imaging from charge state +2 of three endogenous peptides consisting of between 1 and 16 amino acid residues from the acetylated N-terminal end of myelin basic protein: mass-to-charge (m/z) 795.81 (+2) molecular weight (MW) 1589.6, m/z 831.35 (+2) MW 1660.7, and m/z 917.40 (+2) MW 1832.8.
Project description:Atmospheric pressure MALDI on a Q-Exactive instrument was optimized for in-source decay and pseudo-MS3. The dependence of AP-MALDI ISD on the MALDI liquid matrix was investigated for peptides and proteins. The liquid matrices enabled long-life ISD signal, and exhibited high fragment ion yield and signal stability. Extensive a-, b-, c-, y-, and z-type fragment series were observed depending on the matrix used but were most extensive with 2,5-DHB. Complete sequence coverage of small peptide and intact protein-terminus sequence tags were obtained and confirmed using HCD as a pseudo-MS3 method. Graphical Abstract ?.
Project description:RationaleLiquid atmospheric pressure matrix-assisted laser desorption/ionisation (AP-MALDI) has been shown to enable the production of electrospray ionisation (ESI)-like multiply charged analyte ions with little sample consumption and long-lasting, robust ion yield for sensitive analysis by mass spectrometry (MS). Previous reports have focused on positive ion production. Here, we report an initial optimisation of liquid AP-MALDI for ESI-like negative ion production and its application to the analysis of peptides/proteins, DNA and lipids.MethodsThe instrumentation employed for this study is identical to that of earlier liquid AP-MALDI MS studies for positive analyte ion production with a simple non-commercial AP ion source that is attached to a Waters Synapt G2-Si mass spectrometer and incorporates a heated ion transfer tube. The preparation of liquid MALDI matrices is similar to positive ion mode analysis but has been adjusted for negative ion mode by changing the chromophore to 3-aminoquinoline and 9-aminoacridine for further improvements.ResultsFor DNA, liquid AP-MALDI MS analysis benefited from switching to 9-aminoacridine-based MALDI samples and the negative ion mode, increasing the number of charges by up to a factor of 2 and the analyte ion signal intensities by more than 10-fold compared with the positive ion mode. The limit of detection was recorded at around 10?fmol for ATGCAT. For lipids, negative ion mode analysis provided a fully orthogonal set of detected lipids.ConclusionsNegative ion mode is a sensitive alternative to positive ion mode in liquid AP-MALDI MS analysis. In particular, the analysis of lipids and DNA benefited from the complementarity of the detected lipid species and the vastly greater DNA ion signal intensities in negative ion mode.
Project description:The detection of multiply charged helium droplet anions is reported for the first time. By ionizing droplets of superfluid helium with low energy electrons (up to 25 eV), it was possible to produce droplets containing up to five negative charges, which remain intact on the timescale of the experiment. The appearance sizes for different charge states are determined and are found to be orders of magnitude larger than for the equivalent cationic droplets, starting at 4 million He atoms for dianions. Droplets with He*- as charge carriers show signs of being metastable, but this effect is quenched by the pickup of water molecules.
Project description:Ambient ionization mass spectrometry imaging (MSI) has been increasingly used to investigate the molecular distribution of biological tissue samples. Here, we report the integration and optimization of desorption electrospray ionization (DESI) and liquid-microjunction surface sampling probe (LMJ-SSP) with a chip-based high-field asymmetric waveform ion mobility spectrometry (FAIMS) device to image metabolites, lipids, and proteins in biological tissue samples. Optimized FAIMS parameters for specific molecular classes enabled semitargeted detection of multiply charged molecular species at enhanced signal-to-noise ratios (S/N), improved visualization of spatial distributions, and, most importantly, allowed detection of species which were unseen by ambient ionization MSI alone. Under static DESI-FAIMS conditions selected for transmission of doubly charged cardiolipins (CL), for example, detection of 71 different CL species was achieved in rat brain, 23 of which were not observed by DESI alone. Diagnostic CL were imaged in a human thyroid tumor sample with reduced interference of isobaric species. LMJ-SSP-FAIMS enabled detection of 84 multiply charged protein ions in rat brain tissue, 66 of which were exclusive to this approach. Spatial visualization of proteins in substructures of rat brain, and in human ovarian cancerous, necrotic, and normal tissues was achieved. Our results indicate that integration of FAIMS with ambient ionization MS allows improved detection and imaging of selected molecular species. We show that this methodology is valuable in biomedical applications of MSI for detection of multiply charged lipids and proteins from biological tissues.
Project description:We report a novel charge inversion ion/ion reaction that converts multiply charged protein cations to multiply charged protein anions via a single ion/ion collision using highly charged anions derived from nanoelectrospray ionization (nESI) of hyaluronic acids (HAs). This type of charge inversion reaction is demonstrated with cations derived from cytochrome c, apo-myoglobin, and carbonic anhydrase (CA) cations. For example, the reaction has been demonstrated to convert the [CA+22H]22+ carbonic anhydrase cation to a distribution of anions as high in absolute charge as [CA-19H]19-. Ion/ion reactions involving multiply charged ions of opposite polarity have previously been observed to result predominantly in the attachment of the reactant ions. All mechanisms for ion/ion charge inversion involving low energy ions proceed via the formation of a long-lived complex. Factors that underlie the charge inversion of protein cations to high anionic charge states in reaction with HA anions are hypothesized to include: (i) the relatively high charge densities of the HA anions that facilitate the extraction of multiple protons from the protein leading to multiply charged protein anions, (ii) the relatively high sum of absolute charges of the reactants that leads to high initial energies in the ion/ion complex, and (iii) the relatively high charge of the ion/ion complex following the multiple proton transfers that tends to destabilize the complex.
Project description:Detailed understanding of ionic behavior in the region near a charged surface is important for the enhancement of water filtration mechanisms. In this study, a highly charged membrane is hypothesized to form an ion depletion zone (IDZ) without an external power supply. The formation of IDZ was experimentally investigated using membranes with varying surface zeta potential (SZP) values to confirm the hypothesis. The surface zeta potential of the charged membrane was controlled by layer-by-layer deposition method. Our results indicate that indicated that the fluorescent intensity near the charged surface becomes weaker with an increased absolute magnitude of SZP. Ionic surfactants enhance the formation of IDZ by increasing SZP magnitude, and by forming a secondary filtration layer. These findings provide information that is helpful in understanding the ionic behavior near the highly charged surface. In addition, the information provided by the findings would be helpful in fabricating a small-scale water filtration device.
Project description:A novel gas-phase charge and mass manipulation approach is demonstrated to facilitate the mass measurement of high mass complexes within the context of native mass spectrometry. Electrospray ionization applied to solutions generated under native or near-native conditions has been demonstrated to be capable of preserving biologically relevant complexes into the gas phase as multiply charged ions suitable for mass spectrometric analysis. However, charge state distributions tend to be narrow and extensive salt adduction, heterogeneity, and so on tend to lead to significantly broadened peaks. These issues can compromise mass measurement of high mass bio-complexes, particularly when charge states are not clearly resolved. In this work, we show that the attachment of high mass ions of known mass and charge to populations of ions of interest can lead to well-separated signals that can yield confident charge state and mass assignments from otherwise poorly resolved signals.
Project description:Hydrogen exchange in solution combined with ion fragmentation in the gas phase followed by MS detection emerged in recent years as a powerful tool to study higher order protein structure and dynamics. However, a certain type of ion chemistry in the gas phase, namely, internal rearrangement of labile hydrogen atoms (the so-called hydrogen scrambling), is often cited as a factor limiting the utility of this experimental technique. Although several studies have been carried out to elucidate the roles played by various factors in the occurrence and the extent of hydrogen scrambling, there is still no consensus as to what experimental protocol should be followed to avoid or minimize it. In this study we employ fragmentation of mass-selected subpopulations of protein ions to assess the extent of internal proton mobility prior to dissociation. A unique advantage of tandem MS is that it not only provides a means to map the deuterium content of protein ions whose overall levels of isotope incorporation can be precisely defined by controlling the mass selection window, but also correlates this spatial isotope distribution with such global characteristic as the protein ion charge state. Hydrogen scrambling does not occur when the charge state of the precursor protein ions selected for fragmentation is high. Fragment ions derived from both N- and C-terminal parts of the protein are equally unaffected by scrambling. However, spatial distribution of deuterium atoms obtained by fragmenting low-charge-density protein ions is consistent with a very high degree of scrambling prior to the dissociation events. The extent of hydrogen scrambling is also high when multistage fragmentation is used to probe deuterium incorporation locally. Taken together, the experimental results provide a coherent picture of intramolecular processes occurring prior to the dissociation event and provide guidance for the design of experiments whose outcome is unaffected by hydrogen scrambling.