Journal of the American Society for Mass Spectrometry最新文献

Fingerprints are routinely used as evidence in forensic investigations. Fingermarks, any mark left by a donor whether a complete print or not, include sweat and oil excreted by the donor. The chemical components of fingermarks are typically analyzed by gas chromatography–mass spectrometry (GC-MS). Complexity from the number of endogenous and exogenous components associated with fingermarks tends to cause challenging coelutions in resulting chromatograms. In these scenarios, nontargeted analysis can provide substantial benefits over traditional targeted methods that exist in the literature. In this proof-of-concept study, a nontargeted method for analyzing fingermarks was developed and optimized using comprehensive two-dimensional gas chromatography (GC×GC-TOFMS). Two different methods for extracting fingermarks off a microscope slide were evaluated for reproducibility and the quantity of extracted analytes, and a cotton swab collection with solvent extraction was chosen. Instrumental parameters were experimentally optimized to produce a final workflow. The optimized extraction and instrument methods together identified 70 fingermark analytes. Exogenous components within the deposited residue were resolved from endogenous fingermark compounds and used to differentiate donors based on personal care products used by the donor. The extra chromatographic space from GC×GC-TOFMS analysis was beneficial for resolving cosmetic compounds from endogenous fingermark compounds, some of which have been shown to coelute in GC-MS studies previously. The potential for a nontargeted screening of fingermarks for exogenous compounds in a forensic setting is demonstrated as an analysis of trace evidence.

An ongoing goal of top-down mass spectrometry is to increase the performance for larger proteins. Using higher energy activation methods, like 193 nm ultraviolet photodissociation (UVPD), offers the potential to cause more extensive fragmentation of large proteins and thereby yield greater sequence coverage. Obtaining high sequence coverage requires confident identification and assignment of fragment ions, and this process is hampered by spectral congestion and low signal-to-noise ratio (S/N) of the fragment ions. Here we explore the use of charge reduction methods to produce lower charge states of large proteins to increase ion accumulation and generate lower charge states of fragment ions, ones that are naturally dispersed in the m/z domain to alleviate spectral congestion. UVPD of low charge states of enolase (47 kDa) and PRN-1 (63 kDa) resulted in sequence coverages as high as 47% (24+ charge state of enolase) and 23% (32+ of PRN-1) in comparison to 17% (55+ charge state of enolase) and 9% (55+ charge state of PRN-1) obtained for standard high charge states. Proton transfer charge reduction (PTCR) reactions were performed to further disperse fragment ions in the m/z domain and enhance identification. When employing PTCR after UVPD, sequence coverage was maximized for the highest charge states for enolase (55+, 67%) and PRN-1 (55+, 34%), confirming that charge reduction of fragment ions had a more notable impact on outcomes than charge reduction of precursor ions. Sequence coverages were increased further by combining results from electron transfer higher energy collision dissociation (EThcD) and UVPD (85% coverage for enolase and 52% coverage for PRN-1), bolstering the use of complementary MS/MS methods to yield greater dividends for top-down analysis of larger proteins.

The escalating prevalence and diversity of fentanyl analogues poses an immediate concern for the global community. Fentanyl and its analogues are the primary contributors to both fatal and nonfatal overdoses in the United States. The most recent instances of fentanyl-related overdoses have been attributed to the illicit production of fentanyl, characterized by its exceptionally potent nature. In this study, we present a high-throughput mass spectrometry based method for effective screening of fentanyl analogues with focus on the isomeric separation using commercially available platforms combining liquid chromatography, trapped ion mobility spectrometry, and tandem mass spectrometry (LC-TIMS-q-TOF MS/MS). The proposed analysis allows for effective separation and identification of 250 synthetic opioids based on the isotopic pattern, retention time, mobility profile, and MS/MS pattern. Our approach capitalizes on the advancements incorporating parallel accumulation in the mobility trap followed by sequential fragmentation (PASEF) using collision-induced dissociation on the liquid chromatography time scale. While a single chromatography band is commonly observed for single isomeric analogues, a dual mobility band profile attributed to two protonation sites is commonly observed for most fentanyl analogues. Reference mobility values are reported from single standards with 0.2% RSD collected at high resolution (R_IMS ≈ 80–120). The added mobility separation resulted in the separation of isomeric compounds without compromising the sensitivity of the LC-q-TOF MS/MS analysis; that is, a good linear dynamic and (R² > 0.98) and low limits of detection (LOD) in the 0.08–4 ng/mL range were observed for all synthetic analogues (∼100 analogues can be observed with LOD < 1 ng/mL).

In charge detection mass spectrometry (CD-MS) ions are trapped in an electrostatic linear ion trap (ELIT) where they oscillate back and forth through a conducting cylinder. The oscillating ions induce a periodic charge separation that is detected by a charge sensitive amplifier (CSA) connected to the cylinder. The resulting time domain signal is analyzed using short-time Fourier transforms to give the mass-to-charge ratio and charge for each ion, which are then multiplied to give the mass. For ions to be assigned to the correct integer charge states with a low error rate, the charge should be measured with a precision of <0.2 e (elementary charges). Electrical noise reduces the precision of the charge measurement. However, the effect of the noise can be ameliorated by signal averaging, and the measurement time can, in principle, be increased to achieve a precision of <0.2 e. Previously, through optimized ELIT design and improvements to the CSA, the measurement time (with a cryogenically cooled input JFET) required to achieve a charge precision of <0.2 e was reduced by a factor of 2, from 3 s to 1.5 s. In this study, further improvements in JFET selection, capacitance matching, and cryogenic cooling has allowed us to further reduce the electrical noise so that the target precision of <0.2 e can now be achieved for mAb MSQC4 in 600–700 ms with the input JFET cryogenically cooled, and in 900–1000 ms with the input JFET at room temperature. This performance upgrade cuts the overall time for high-resolution charge measurements by more than another factor of 2. For a measurement time of 100 ms, the charge RMSD is 0.51 e with cryogenic cooling. The results presented here further cements CD-MS with an ELIT as the fastest and most accurate approach to single ion MS measurements.

An integrated miniature time-of-flight mass spectrometer (TOF-MS) system coupled with a pocket-size 3D-printed laser-induced acoustic desorption (LIAD) source is described. This 3D-printed LIAD source utilizes only a miniature deceleration motor to achieve two-dimensional motion of the target surface, simplifying the source structure and improving the long-term stability of mass spectrometry measurements. It has been successfully applied to analyze the model molecule creatinine and ingredients in an energy beverage (Red Bull), where main natural nutrients were clearly identified. By adjusting the 3D printing parameters, the source can be readily adapted to various application scenarios requiring different sizes. This work provides a simple and efficient 3D-printed source injection strategy for subsequent investigations in molecular reaction dynamics.

This study explores the computational isolation of prostaglandin (PG) isomers, specifically PG E₂ (PGE₂) and D₂ (PGD₂), to enhance method development efficiency and provide insights into their retention behavior during supercritical fluid extraction (SFE) combined with supercritical fluid chromatography (SFC)-tandem mass spectrometry (MS/MS). Although PGE₂ and PGD₂ are positional isomers that yield identical product ions in MS/MS, they serve distinct biological roles. This research illustrates the efficacy of selected reaction monitoring (SRM)-based techniques for differentiating coeluting isomers. Despite the challenges posed by baseline resolution, simplified computational methods successfully distinguished between PGE₂ and PGD₂, demonstrating the potential for high-throughput PG analysis without the necessity for complete chromatographic peak resolution. By employing least-squares estimation to solve a linear system, the abundance ratio of PGE₂ to PGD₂ was derived from intensity ratios across four SRM transitions, achieving precise quantification even with poorly resolved SFC peaks. The study highlights critical factors affecting PG retention, such as the choice of the stationary phase, temperature regulation, and reduction of stainless steel interactions, which can diminish signal intensity. A significant observation is the concentration-dependent suppression effect of the entrainer when interacting with the hepatocyte matrix, underscoring the importance of effective matrix management in SFE/SFC-MS/MS. These findings advance the development of a robust, high-throughput analytical platform for PG quantification and lipidomics research applications.

Electrospray ionization (ESI)-mass spectrometry (MS) is a key platform for analyzing post-translationally modified proteins. With continuous advances in MS instruments and data analysis methods, top-down analysis of intact proteoforms has become highly feasible. To accurately quantify proteoforms with varying post-translational modifications (PTMs), the influence of PTMs on the ESI-MS detection efficiency must be considered. Two decades ago, Kelleher and co-workers proposed using protein ion relative ratios (PIRRs) and fragment ion relative ratios (FIRRs) in ESI-MS for proteoform quantification. While FIRR quantification has been extensively studied, the reliability of PIRR quantification─particularly for proteoforms with varying PTM degrees─remains under-evaluated. In this study, we further validated the fidelity of PIRR quantification in top-down ESI-MS using various site-specifically acetylated recombinant histone H3 proteoforms. These proteoforms, carrying varied degrees of acetylation, were produced using an orthogonal translation system that incorporates acetyllysine at the amber stop codons. After absolute quantification by UV spectrophotometry, samples were mixed in isometric ratios and analyzed by either direct infusion-ESI-MS or weak cation exchange/hydrophilic interaction-ESI-MS. Our results show that PIRRs match theoretical ratios regardless of the acetylation degree or site. These findings reinforce the validity of top-down proteoform quantification, especially for histone proteins.

Zhongzi purple rice is recognized as a nutritionally superior whole-grain variety, containing higher levels of protein, iron, dietary fiber, and vitamin B6 compared to conventional rice. While the nutritional profile of Zhongzi purple rice is well-established, the spatial distribution and structural specificity of its lipid components, especially germ-specific triacylglycerols (TAGs), remain poorly characterized. This study employs a multimodal mass spectrometric strategy to investigate the lipidomic uniqueness of the Zhongzi purple rice. The strategy combines matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for spatial analysis, high-resolution mass spectrometry (HRMS) for primary analysis, and high-resolution tandem mass spectrometry with atmospheric pressure chemical ionization (APCI-HRMS/MS) for secondary analysis. MALDI-MSI identified seven metabolites in Zhongzi purple rice that exhibited germ-specific accumulation. In contrast, these metabolites showed markedly reduced signal intensities and lacked structured spatial distribution in conventional purple, black, and brown rice. Using a combination of lipidomic database interrogation, HRMS molecular formula validation, and APCI-HRMS/MS structural elucidation, we identified these metabolites as unsaturated fatty acid-enriched TAGs. Specifically, they were TAG(16:0/16:0/18:2), TAG(16:0/16:0/18:1), TAG(16:0/18:1/18:3), TAG(16:0/18:1/18:2), TAG(16:0/18:1/18:1), TAG(18:1/18:2/18:2), and TAG(18:1/18:1/18:2). The high unsaturation of these TAGs contributes to improved lipid hydrolysis efficiency, metabolic health benefits, and oxidative stability. These properties highlight Zhongzi purple rice as a potential functional food source. Due to their germ-specific accumulation, these compounds can act as potential biomarkers for authenticating Zhongzi purple rice and detecting adulterants. This provides a practical solution to commercial challenges in the industry. This study pushes the boundaries of cereal lipidomics through the integration of spatially resolved imaging and structural validation. The results shed light on the role of lipids in driving the nutritional and metabolic benefits of whole grains.

Quantifying the similarity between two mass spectra─a known reference mass spectrum and an unidentified sample mass spectrum─is at the heart of compound identification workflows in gas chromatography–mass spectrometry (GC-MS). The reference spectrum most like the sample is assigned as its identification (provided some quantitative similarity threshold is met, e.g., 80%) and thus accurately measuring similarity is essential. Significant research has gone toward developing metrics for this purpose, each of which has attempted to improve upon existing methods by incorporating GC-MS-specific information (e.g., peak ratios or retention times) or adopting various statistical and algorithmic frameworks. While this active development has led to a plethora of similarity metrics with demonstrated value across different contexts, the unfortunate consequence has been confusion surrounding which metric should be used as a global standard. No such metric is currently accepted as the standard method because different metrics have demonstrated optimal performance in different contexts. In this work, we propose an ensemble approach to spectral similarity scoring that combines the collective information from across existing similarity metrics to form an improved, globally representative similarity metric as a step toward establishing a global standard method. The resulting ensemble metrics are evaluated on over 88,000 spectra of varying complexity and demonstrate improved abilities to accurately rank the correct reference spectrum as the top-matching candidate for a sample relative to the rankings generated by individual similarity scores.

Neutral lipids are vital to various cellular processes and disease pathologies. However, their characterization by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) remains challenging due to poor ionization efficiency and difficulties distinguishing subtle structural differences among numerous isomeric and isobaric species. In this study, we enhanced neutral lipid detection by incorporating isotonic metal–cation washes into our MALDI IMS sample preparation workflow. Resulting salt adducts improved neutral lipid isobar and isomer separation by using trapped ion mobility spectrometry (TIMS). This approach increased both sensitivity and specificity for neutral lipid IMS experiments across multiple organ types, including murine brain, rabbit adrenal gland, human colon, and human kidney. Comparative analyses revealed that the most effective salt wash was tissue-dependent. However, the Na⁺ carbonate buffer solution (CBS) wash showed the greatest overall increase in neutral lipid detection. These findings provide a robust framework for mapping neutral lipids across multiple tissues and disease states and allow for the detailed characterization of neutral lipid isomers and isobars in complex biological tissues.