Journal of the American Society for Mass Spectrometry最新文献

A miniature ion trap particle mass spectrometer with both optical and charge detection systems was constructed in this work. The instrument was initially constructed with an open architecture and mounted on a 600 × 450 mm optical bench. With further compact integration, the system can be accommodated within an aluminum enclosure with dimensions of 333 × 221 × 192 mm and a total mass of approximately 8 kg. In the optical detection mode, the stationary star pattern ion motion was observed by detecting the scattered light, and the m/Z value of the particle ion was calculated accurately. In the charge detection mode, the particle m/Z value, the charge number Z, and the particle mass were determined quickly. These two working modes can be switched freely. By using 3 μm polystyrene size standards and mice red blood cells as the sample, the feasibility of this instrument was demonstrated.

Fragmentation of protonated threonine isomers was investigated by multiple-stage tandem mass spectrometry (MSⁿ) with ion-trap collision-induced dissociation (CID) and density functional theory (DFT) calculations. Protonated molecules containing oxygen or nitrogen often produce heterocyclic fragments by CID. DFT calculations revealed that H₂O loss from threonine isomers produced cyclic amines, lactams, and lactones. The transition-state barriers and rate constant for the formation of these fragments are highly dependent on the ring size. Although 3-membered cyclic amines are observed in the product ion spectrum, lactams and lactones are formed only as rings larger than four and five members, respectively. H₂O loss from the protonated threonine side chain produced a 3-membered cyclic amine containing a carboxyl group, which undergoes loss of the combined elements of H₂O and CO upon CID. H₂O loss from protonated homoserine and β-homoserine produced 5-membered lactone and 4-membered lactam, respectively. Further dissociation of the corresponding lactone and lactam results in fragment ions formed via the loss of CO and CH₂CO, respectively. In contrast, H₂O loss from the protonated γ-amino-β-hydroxybutyric acid provides 5-membered lactams, which undergo only further H₂O loss upon CID. MS³ analysis of protonated threonine isomers through dehydrated precursor ions produced different fragment ions. The threonine isomers could be distinguished by characteristic fragment ions, and the molar ratio of the isomers in the mixture can be estimated from the relative abundances of their fragment ions. These results demonstrate MSⁿ with ion-trap CID to be a useful method for the identification and semiquantification of threonine isomers.

The time course of metabolic labeling using deuterated water, followed by liquid chromatography coupled with mass spectrometry, is employed to investigate the turnover rates of individual proteins in vivo. These labeling experiments are resource intensive. Computational methods that can determine turnover rates from a single and labeled for a short duration sample will help reduce these demands. We evaluated linear and logarithmic models to estimate protein turnover rates based on two samples (one nonlabeled and one labeled). Key factors such as the number of exchangeable hydrogens, body water enrichment in deuterium, protein turnover rate, and necessary changes in monoisotopic relative abundance established a range of labeling durations for the two-sample approach. We provide two inequalities that formally define this range of labeling duration for each peptide, which is integrated into an R Shiny App. We applied this two-sample approach to four murine tissues. By adjusting the labeling duration according to the turnover of the tissue proteome, the two-sample approach was able to analyze over 60% (1221 murine liver proteins) of the proteome previously assessed using a multisample approach.

Ultraviolet photodissociation (UVPD) of proteins is known to exhibit conformation-dependent fragmentation patterns, but direct structural evidence linking precursor protein and fragment ions has been limited. Here, we apply tandem trapped-ion mobility spectrometry/tandem-mass spectrometry to compare collision cross sections of UVPD fragment ions generated from distinct conformers of ubiquitin. Under the high-pressure (∼4 mbar) and low-photon density (∼10 μJ laser pulse energies) conditions employed here, UVPD produces predominantly [b + 2] and [y - 2] ions at proline residues, consistent with direct bond cleavage from the electronically excited state. Our data show that these ions can retain a clear structural relationship to the precursor conformation: UVPD of compact, native-like ubiquitin yields fragments with collision cross sections ∼20% smaller than the corresponding ions produced from extended precursors or by collision-induced dissociation. Further, these compact UVPD fragments are kinetically trapped in metastable conformations, with substantial barriers preventing relaxation toward energetically favored gas-phase structures. We attribute this behavior to limited vibrational energy deposition per absorbed 213 nm photon combined with rapid collisional cooling, which suppress cumulative thermal activation and disfavor statistical fragmentation pathways, leaving direct excited-state dissociation as the dominant observable process. Together with prior UVPD studies on holo-myoglobin, our results suggest that UVPD fragments can retain aspects of their precursor tertiary structure.

The stability of proteins from the rates of oxidation (SPROX) technique is a mass spectrometry-based approach for making protein folding stability measurements on the proteomic scale. The development and application of SPROX, to date, have primarily relied on the use of quantitative bottom-up proteomics and data-dependent acquisition (DDA) strategies using isobaric mass tags. Use of isobaric mass tags is attractive, as it enables the mass spectrometry readout in SPROX to be highly multiplexed. However, the use of such isobaric mass tags is restricted to DDA strategies, which can be limited in their proteomic coverage compared with data-independent acquisition (DIA) strategies. Reported here is a new "one-pot" SPROX workflow that employs a DIA readout and a label-free quantification strategy. Analysis of the proteins in an E. coli cell lysate using the DIA-SPROX strategy allowed for the calculation of transition midpoints with reasonable accuracy. The proteins from a S. cerevisiae cell lysate were also assessed for ligand-induced changes in their transition midpoints upon the introduction of cyclosporine A (CsA) to identify the protein targets of this well-studied ligand. The DIA-SPROX strategy developed here successfully identified known protein targets of CsA with a low false positive rate using a combination of two different software, Spectronaut and DIA-NN, for DIA data processing. We also find that the proteomic coverage obtained using DIA-SPROX is comparable to the coverage obtained in conventional DDA-SPROX experiments. Significantly, this comparable coverage can be achieved without a fractionation strategy (e.g., methionine-containing peptide enrichment) in DIA-SPROX.

Charge detection mass spectrometry (CD-MS) enables mass measurements to be made for heterogeneous samples into the gigadalton regime. In CD-MS, ions are trapped in an electrostatic linear ion trap (ELIT) where they oscillate back and forth through a detection cylinder. The m/z is determined from the oscillation frequency, and the charge is obtained from the signal amplitude. The charge can be measured with a precision of better than 0.2 e (elementary charges), where ions can be assigned to the correct charge state with a low error rate. Thus, the main factor limiting mass resolution in CD-MS measurements is the imprecision in the m/z determination for individual ions. In prior work, it was shown that m/z resolving powers >300,000 could be achieved by optimizing the ELIT design to minimize the dependence of the ion's oscillation frequency on the ion's kinetic energy and trajectory. However, the high-resolution ELIT designs that we found were intolerant to small misalignments of the trap electrodes that result from manufacturing imprecision. A misalignment of less than 20 μm caused the trapping efficiency to drop to zero. The best resolving power achieved with a more tolerant ELIT design (where manufacturing imprecision does not catastrophically reduce the trapping efficiency) is 14,000-15,000. Here, we explore a solution to the intolerant ELIT designs where some of the ELIT mirror electrodes are segmented to allow small trim potentials to correct for mechanical misalignments. The trim potentials can be optimized under computer control to maximize trapping efficiency and m/z resolution. Trajectory simulations indicate that a high trapping efficiency can be recovered (>90%) while retaining high resolving powers (>200,000).

2-Butanone peroxide is a commonly used cross-linker in the chemical industry and a homemade explosive encountered by the forensic community. In this study, the dissociation behavior of ammonium cationized MEKP oligomers was explored using tandem MS. The shorter ammoniated MEKP oligomer cations n = 3, 4 underwent charge-induced fragmentation pathways, which generated cyclic MEKP and smaller oligoperoxide fragment ions. Control experiments with metal ion adducts of MEKP oligomers n = 3, 4 generated distinct dissociation behavior and supported the initial pathway assignment. Longer ammoniated MEKP oligomers n = 6-9 underwent charge-remote fragmentation pathways, which generated terminally modified alkyl ketone MEKP fragment ions. The dissociation behavior followed that of metalated MEKP oligomer cations, which predominantly dissociate through charge-remote pathways. The change in the fragmentation pathway from shorter to longer oligomer length indicates that ammonium is more strongly bound in the longer MEKP oligomers and does not participate in bond breaking and formation. Additional MS³ analyses demonstrated that the alkyl ketone MEKP fragment ions readily undergo consecutive fragmentation to generate mono- and diterminal modifications. Ammoniated MEKP oligomers n = 5 exhibited a combination of charge-induced and -remote pathways, representing a cross over point between the two pathways. Results here will aid future efforts in organic peroxide detection and characterization, especially with considerations to ion adducts, fragmentation pathways, fragment ion-types, and MS approaches.

Mass spectrometry imaging (MSI) provides spatially resolved chemical analysis of surfaces and is widely applied in biological and biomedical research. Multimodal MSI can combine techniques such as secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) to leverage their complementary strengths. However, acquiring multimodal MSI data using separate instruments introduces challenges, including image coregistration and potential sample degradation during transfer. To address these limitations, we previously integrated a C₆₀ ion gun into a prototype, commercially available MSI instrument, enabling SIMS, MALDI, and secondary electron imaging within a single platform. In this study, we implemented field of view (FoV) mode SIMS on this platform by rastering the ion beam, achieving an improved spatial resolution of 2 μm and surpassing the spatial resolution of the sample stage. Additionally, we optimized the instrument for elemental ion signals and observed enhanced sensitivity of selected species in SIMS through collision-induced dissociation (CID). To explore the usability of the ion gun for high-spatial-resolution image coregistration with other imaging modalities, fiducial markers were etched using the ion gun, creating a localized absence of signal in the etching locations, which can aid coregistration with optical imaging. Additionally, the secondary electrons produced by rastering the ion beam were used to image and assess the MALDI laser focus and power, allowing us to determine the optimal settings.