Journal of the American Society for Mass Spectrometry最新文献

Aliphatic hydrocarbons and hydrocarbon-based synthetic polymers are of interest in many fields, but their characterization by mass spectrometric methods is generally limited due to their poor ionizability. Recently, atmospheric pressure photoionization (APPI), combined with halogen anion attachment in negative-ion mode, has drawn attention as a potential method for ionizing various polymers without extensive fragmentation or other unwanted side reactions. In this work, the applicability of halogen anion attachment with APPI was studied using several synthetic polymers, including polyethylene, polypropylene, polyisoprene, and polystyrene, as well as simple n-alkanes of various chain lengths. For hydrocarbon-based polymers, the method produced clear distributions of intact polymer adduct ions when different halogen anions were used. It was found that increasing the halogen anion size decreased ionization efficiency, particularly in the absence of π-bonds in the polymer structure. Testing with simple n-alkanes showed that only molecules containing fifty or more carbon atoms formed detectable halogen adducts, possibly due to the low gas-phase stabilities of the lighter n-alkane adduct ions. In conclusion, halogen anion attachment with negative-ion APPI appears to be a highly promising method for polymer analysis, providing structural data and clean polymer mass spectra with minimal fragmentation, which can be useful for the identification of unknown samples.

This Perspective covers discovery and mechanistic aspects as well as initial applications of novel ionization processes for use in mass spectrometry that guided us in a series of subsequent discoveries, instrument developments, and commercialization. Vacuum matrix-assisted ionization on an intermediate pressure matrix-assisted laser desorption/ionization source without the use of a laser, high voltages, or any other added energy was simply unbelievable, at first. Individually and as a whole, the various discoveries and inventions started to paint, inter alia, an exciting new picture and outlook in mass spectrometry from which key developments grew that were at the time unimaginable, and continue to surprise us in its simplistic preeminence. We, and others, have demonstrated exceptional analytical utility. Our current research is focused on how best to understand, improve, and use these novel ionization processes through dedicated platforms and source developments. These ionization processes convert volatile and nonvolatile compounds from solid or liquid matrixes into gas-phase ions for analysis by mass spectrometry using, e.g., mass-selected fragmentation and ion mobility spectrometry to provide accurate, and sometimes improved, mass and drift time resolution. The combination of research and discoveries demonstrated multiple advantages of the new ionization processes and established the basis of the successes that lead to the Biemann Medal and this Perspective. How the new ionization processes relate to traditional ionization is also presented, as well as how these technologies can be utilized in tandem through instrument modification and implementation to increase coverage of complex materials through complementary strengths.

We introduce Fluorescence Integrated Single-Cell Analysis Script (FISCAS), which combines fluorescence microscopy with MALDI-MSI to streamline single-cell analysis. FISCAS enables automated selection of tight measurement regions, thereby reducing the acquisition of off-target pixels, and makes use of established algorithms for cell segmentation and coregistration to rapidly compile single-cell spectra. MALDI-compatible staining of membranes, nuclei, and lipid droplets allows the collection of fluorescence data prior to the MALDI-MSI measurement on a timsTOF fleX MALDI-2. Usefulness of the software is demonstrated by the example of THP-1 cells during stimulated differentiation into macrophages at different time points. In this proof-of-principle study, FISCAS was used to automatically generate single-cell mass spectra along with a wide range of morphometric parameters for a total number of roughly 1300 cells collected at 24, 48, and 72 h after the onset of stimulation. Data analysis of the combined morphometric and single-cell mass spectrometry data shows significant molecular heterogeneity within the cell population at each time point, indicating an independent differentiation of each individual cell rather than a synchronized mechanism. Here, the grouping of cells based on their molecular phenotype revealed an overall clearer distinction of the different phases of differentiation into macrophages and delivered an increased number of lipid signals as possible markers compared with traditional bulk analysis. Utilizing the linkage between mass spectrometric data and fluorescence microscopy confirmed the expected positive correlation between lipid droplet staining and the overall signal for triacylglyceride (TG), demonstrating the usefulness of this multimodal approach.

Theta emitters are useful for generating microdroplets for rapid-mixing reactions. Theta emitters are glass tips containing an internal septum that separates two channels. When used for mixing, the solutions from each channel are sprayed with mixing occurring during electrospray ionization (ESI) with reaction times on the order of microseconds to milliseconds. Theta emitters of increasing size cause the formation of ESI droplets of increasing size, which require longer times for desolvation and increase droplet lifetimes. Droplets with longer lifetimes provide more time for mixing and allow for increased reaction times prior to desolvation. Because theta emitters are typically produced in-house, there is a need to consistently pull tips with a variety of sizes. Herein, we characterize the effect of pull parameters on the generation of distinct-sized theta emitters using a P-1000 tip puller. Of the examined parameters, the velocity value had the largest impact on the channel diameter. This work also compares the effect of pulling parameters between single-channel and theta capillaries to examine how the internal septum in theta capillaries affects tip pulling. We demonstrate the utility of using theta emitters with different sizes for establishing distinct reaction times. Finally, we offer suggestions on producing theta emitters of various sizes while maintaining high repeatability. Through this work, we provide resources to establish a versatile and inexpensive rapid-mixing system for probing biologically relevant systems and performing rapid derivatizations.

Mass spectrometry is a powerful analytical technique used at every stage of the pharmaceutical research process. A specialized branch of this method, mass spectrometry imaging (MSI), has emerged as an important tool for determining the spatial distribution of drugs in biological samples. Despite the importance of MSI, its quantitative capabilities are still limited due to the complexity of biological samples and the lack of separation prior to analysis. This makes the simultaneous quantification and visualization of analytes challenging. Several techniques have been developed to address this challenge and enable quantitative MSI. One such approach is the mimetic tissue model, which involves the incorporation of an analyte of interest into tissue homogenates at several concentrations. A calibration curve that accounts for signal suppression by the complex biological matrix is then created by measuring the signal of the analyte in the series of tissue homogenates. Herein, we use the mimetic tissue model on a triple quadrupole mass spectrometer (QqQ) in multiple reaction monitoring mode to demonstrate the quantitative abilities of nanospray desorption electrospray ionization (nano-DESI) and compare these results with those obtained using atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI). For the tested compounds, our findings indicate that nano-DESI achieves lower standard deviations than AP-MALDI, resulting in superior limits of detection for the studied analytes. Additionally, we discuss the limitations of the mimetic tissue model in the quantification of certain analytes and the challenges involved with the implementation of the model.

We present ion dynamics simulations regarding the effect of space charge on the performance of Fourier transform quadrupole ion traps (FT-QITs) with special attention to signal stability, mass resolving power, and sensitivity. Ion trajectory simulations within an idealized QIT geometry are performed by applying a dedicated application (QITSim) using an in-house developed open simulation framework (IDSimF). Image current detection transients are generated by the application and are subsequently transformed into frequency spectra of ion secular motion. Such frequency spectra are the basis for the calculation of mass spectra in FT-QIT instruments. The simulation results are used to assess the extent of space charge induced effects regarding the analytical performance of FT-QIT systems. The simulation results for two Cl⁺ and seven Xe⁺ isotope ions exhibit diverse space charge induced phenomena. Most prominently, complete isotope signal fusion, quantitative individual signal suppression, and severe signal distortions are observed even at low absolute numbers of trapped ions. Furthermore, significant shifts of the secular oscillation frequencies occur, even when the signal shape appears to be mostly unaffected and when the frequency separation for trapped ions with different masses is large. This significantly limits the applicability of FT-QIT systems for high resolution mass spectrometry. An increase of the RF amplitude of the trapping field as well as increasing the extent of ion excitation partly mitigate these adverse space charge effects; nevertheless, the usable operational range of the simulated FT-QIT system for analytical applications remains rather narrow.

HNTX-XXI, a peptide toxin derived from the venom of the spider Ornithoctonus hainana, comprises a 64-amino-acid protein architecture that notably incorporates eight cysteine residues positioned at positions 2, 10, 14, 16, 17, 23, 36, and 63. The close spatial proximity of Cys16 and Cys17 poses a challenge in resolving their disulfide bridge configurations using standard methodologies. In this study, we introduce an innovative and highly efficient approach for delineating disulfide pairings in peptides containing adjacent cysteines. Our methodology integrates a two-step proteolytic digestion strategy utilizing trypsin and Glu-specific staphylococcal V8 protease coupled with a subsequent round of Edman degradation. This multifaceted approach enables the precise characterization of the disulfide bonds within the peptide. Specifically, targeted proteolysis by trypsin and V8, followed by reversed-phase HPLC separation of the resulting peptides, facilitated the unambiguous identification of disulfide linkages between Cys10-Cys23 and Cys14-Cys63. For the fragment containing the four remaining cysteines, a single cycle of Edman degradation was employed, strategically breaking the peptide bond between the adjacent cysteines. This pivotal step enabled the isolation and analysis of the resulting fragments. Subsequently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was utilized, revealing the presence of two additional disulfide bonds: Cys2-Cys17 and Cys16-Cys36. Collectively, these findings allow for the definitive assignment of the four disulfide linkages in HNTX-XXI as Cys2-Cys17, Cys10-Cys23, Cys14-Cys63, and Cys16-Cys36. This rapid and sensitive methodology represents a significant advancement in the structural characterization of peptide toxins with complex disulfide bond patterns, underscoring its potential for broad application in the field of venom peptide research.

In this study, we evaluate lipids and select proteins in human lung fibroblasts (hLFs) to interrogate changes occurring due to aging and senescence. To study single cell populations, a comparison of cells adhered onto slides using poly-d-lysine versus centrifugal force deposition was first analyzed to determine whether specific alterations were observed between preparations. The poly-d-lysine approach was then utilized to interrogate the lipidome of the cell populations and further evaluate potential applications of the MALDI-immunohistochemistry (IHC) platform for single-cell-level analyses. Two protein markers of senescence, vimentin and p21, were both observed within the fibroblast populations and quantified. Lipidomic analysis of the fibroblasts found 12 lipids significantly altered because of replicative senescence, including fatty acids, such as stearic acid, and ceramide phosphoethanolamine species (CerPE). Similar to previous reports, alterations were detected in putative fatty acid building blocks, ceramides, among other lipid species. Altogether, our results reveal the ability to detect lipids implicated in senescence and show alterations to protein expression between normal and senescent fibroblast populations, including differences between young and aged cells. This report is the first time that the MALDI-IHC system has been utilized at a single-cell level to analyze both protein expression and lipid profiles in cultured cells, with a particular focus on changes associated with aging and senescence.