Journal of the American Society for Mass Spectrometry最新文献

Top-down mass spectrometry (MS) enables comprehensive characterization of modified proteins and nucleic acids and, when native electrospray ionization (ESI) is used, binding site mapping of their complexes with native or therapeutic ligands. However, the high complexity of top-down MS spectra poses a serious challenge to both manual and automated data interpretation, even when the protein, RNA, or DNA sequence and the type of modification or the ligand are known. Here, we introduce FAST MS, a user-friendly software that identifies, assigns and relatively quantifies signals of molecular and fragment ions in MS and MS/MS spectra of biopolymers with known sequence and provides a toolbox for statistical analysis. FAST MS searches mass spectra for ion signals by comparing all signals in the spectrum with isotopic profiles calculated from known sequences, resulting in superior sensitivity and an increased number of assigned fragment ions compared to algorithms that rely on artificial monomer units while maintaining the false positive rate on a moderate level (<5%). FAST MS is an open-source, cross-platform software for the accurate identification, localization and relative quantification of modifications, even in complex mixtures of positional isomers of proteins, oligonucleotides, or any other user-defined linear polymer.

Fluorescence labeled glycan homologous mixtures were quantified using fluorescence and then used to evaluate ionization performances in electrospray ionization at micro, nano, and femto flow modes. nanoESI produced higher (2+ and 3+) charged ions adducted with sodium and calcium. In comparison, femtoESI was found to favor the generation of [M + H]⁺ ions against metal adducts, even with nonvolatile salts up to 1 mM for NaCl and 100 μM for CaCl₂. For labeled glucose homopolymer (GHP) glycans, nanoESI and femtoESI had 0.81 and 3 nM detection limits, respectively. With LC separation and a much higher flow rate, conventional microflow ESI detected all glycans with 10-fold lower concentrations. Overall, nanoESI had the optimum uniformity in the relative ionization efficiency (RIE). When summing up intensities of analyte ions formed with all charge carriers, the RIE of the midsized glycans (10 to 16 glucose units) appear to be uniform (RIE 95%-105%). For the smaller (1-5 glucose units) glycan components, femtoESI provided better uniformity than nanoESI and conventional ESI. For the labeled IgG N-glycans, the impact of chemical structure on the ionization efficiency was revealed by the strong correlation between their RIE trends in different ionization modes.

Tandem mass spectrometry (MSⁿ) is one of the most effective methods to obtain the structures of organic molecules, enabling the observation of multigenerational ion fragments. Collision-induced dissociation (CID) is currently the most mature technique for mass spectrometry analysis. Ion trap mass spectrometry (ITMS) is favored for on-site detection field, due to its ability of MSⁿ analysis with a single trap and its small size. However, conventional MSⁿ analysis in ITMS requires repeated isolation and excitation processes multiple times, causing high complexity of the entire scanning process. Additionally, the fragment ion detection in ITMS is limited by low-mass cutoff (LMCO) and the weak fragmentation yield. In this study, a method named reverse scanning-collision induced dissociation (RS-CID) is proposed, which involves increasing RF and AC frequencies while maintaining RF voltage constant during the CID process. Twelve representative illegal drugs were analyzed adopting this method, enhancing the intensities of low-mass fragment ions compared to conventional dissociation method. Moreover, experimental results with ketamine and methamphetamine show that RS-CID effectively reduces the LMCO effect and slightly improves CID efficiency. It also enables direct acquisition of their multigeneration fragment ions spectra in a single sequence of ion injection, cooling, isolation, RS-CID, cooling, mass scanning and empty. The experiments to distinguish between the isomers ab-4en-pinaca and adb-3en-butinaca as well as the isomeric compounds 5f-cumyl-pegaclone and cumyl-pipetinaca were also successful by this method. In summary, RS-CID enables MSⁿ analysis in a single sequence and improves the low-mass fragment ions intensity. It can simplify workflows, achieve faster analysis and provide more valuable mass spectral information.

Sample carryover is a common problem in hydrogen-deuterium exchange mass spectrometry, particularly because immobilized protease columns cannot withstand the high organic solvent concentrations typically used in liquid chromatography-mass spectrometry (LC-MS) for cleaning. Conventional cleaning methods using injections of guanidine HCl still suffer from carryover and may require four blanks after each sample run to fully remove carryover. We have implemented an additional LC pump to deliver customized wash solutions to protease and enzyme columns, and the associated LC capillaries to eliminate carryover. Pump-based washing using the protease-safe wash solutions tested herein was able to fully remove carryover with only one blank run. FOS-choline-12 was found to be the most effective component in wash solutions and even performed well alone at 0.1% volume concentration. Since the protease column washing is performed concurrently during the analytical gradient within a sample run, subsequent blank runs could be reduced from four to one and total run time could be reduced by up to 60%. Savings in total run time could more than double the productivity of data acquisition, which is imperative for pandemic preparedness and for acceleration of biotherapeutics development.

Sodium dodecyl sulfate (SDS) plays a pivotal role in protein denaturation, tissue extraction, and protein mass-based electrophoretic separations. However, even modest concentrations of SDS can cause column overpressure, retention time shifts, and ionization signal suppression during liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Thus, SDS removal is a critical step for LC-MS/MS analysis of protein digests containing SDS. This study describes an inexpensive and high-throughput method to remove SDS from protein digests using weak-anion exchange (WAX) resins in 96-well filter plates. Requiring less than 3 min, this method can reduce SDS concentrations from 0.1-0.4% to less than 5 ppm and from 0.6-1% to less than 100 ppm. After SDS removal, the recoveries of unmodified tryptic peptides and phosphorylated peptides (at 94.3 nM) were ∼90% and ∼70%, respectively. Additionally, when using aqueous 1% SDS to solubilize trastuzumab-spiked mouse serum and subsequently removing the SDS using the WAX resin, quantitation of trastuzumab exhibited excellent linearity (R² = 0.9996) together with a low coefficient of variation (<10%). Calculated concentrations were within 20% of the expected value for spiked standard samples (0.5, 1, and 2 μg/mL trastuzumab in mouse serum). The method is about 20× more cost-effective versus commercialized SDS removal kits and both the resin and filter plate are readily available, so the method should easily transfer to other laboratories.

When petroleum leaks into soil, the polar compounds exhibit strong biological toxicity, causing serious damage to soil animals, plants, and microorganisms and potentially threatening human health. However, the systematic comprehension of the migration of polar compounds in petroleum-contaminated soil remains limited. Herein, we employed elemental analysis, stable carbon isotope analysis, and high-resolution mass spectrometry techniques to study the migration of polar compounds in petroleum-contaminated soil using a column leaching experiment. The results indicate that petroleum migration ability in soil is limited, and the compounds are primarily concentrated in the soil above 40 cm. The C/N, C/H, and δ¹³C ratios of organic matter in soils are highly affected by petroleum contamination. Meanwhile, the different compound classes show varying migration abilities, with N₁ and N₁O₁ compounds exhibiting stronger adsorption capacity on soil, while oxygen-containing compounds are more likely to migrate with water to deeper soil. Additionally, molecular polarity, unsaturation degree, and size are key factors affecting the migration of polar compounds in petroleum within the soil. This simulation experiment offers valuable insights into comprehending migration of polar compounds in petroleum-contaminated soil and their potential impacts for soil ecological environment.

One of the main challenges in mass spectrometry imaging data analysis remains the analysis of m/z-spectra displaying a low signal-to-noise ratio caused by their low abundance, sample preparation, matrix effects, fragmentation, and other artifacts. Additionally, we observe that molecules with a high abundance suppress those with lower intensities and misdirect classical tools for MSI data analysis, such as principal component analysis. As a result, the observed significance of a molecule may not always be directly related to its abundance. In this work, we present a recursive rank-2 non-negative matrix factorization (rr2-NMF) algorithm that automatically returns spectral and spatial visualization of colocalized molecules, both highly and lowly abundant. Using this hierarchical decomposition, our method finds spatial and spectral correlations on different levels of abundances. The quality of the analysis is evaluated on MALDI-TOF data of healthy mouse pancreatic tissue for the annotation of molecules of interest in the lower abundances. The results show interesting findings regarding the functioning and colocalization of certain molecules.

In this study, a low-cost 4.3 MHz plasma ionization source for ion mobility spectrometry (IMS), utilizing a miniaturized Tesla coil, is presented. This compact design, combined with a 3D printed cyclic olefin copolymer (COC) housing, delivers a stable and directed plasma suitable for ionization in IMS applications. The 3D printed housing ensures chemical resistance and low off-gassing, which are crucial for maintaining sample integrity. The Tesla coil produces a consistent sine wave at 4.3 MHz, and when connected to stainless steel screw electrodes it generates a stable plasma capable of ionizing analytes such as limonene, MTBE, nicotine, 2-octanone, and propofol. Measurements were conducted in both positive and negative ion modes. The results demonstrate the Tesla coil's effectiveness as a low-cost and reliable ionization source for IMS, offering comparable performance to traditional Ni⁶³ β-emitters. This advancement in plasma ionization technology could facilitate more accessible and flexible IMS systems for diverse analytical applications. The integration of 3D printing in the development of this ionization source underscores the potential for customized, low-cost analytical instrumentation, promoting innovation in laboratory environments and commercial applications.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurement data and machine learning were used in this work to classify six different types of plastics. In order to take into account the characteristics of the measurement data, the local maxima of the measurement data were first examined in a preprocessing step. Several machine learning methods were then implemented to create a model that could successfully classify the plastics. To visualize the data distribution, we applied a dimensionality reduction method, namely, principal component analysis. Finally, to distinguish between the six types of plastics, we conducted an ensemble analysis using four tree-based algorithms: decision tree, random forest, gradient boosting, and LIGHTGBM. This approach can identify the feature importance of plastic samples and allow the inference of the chemical properties of each plastic type. In this way, ToF-SIMS data could be utilized to successfully classify plastics and enhance explainability.

Protein lactylation is a novel post-translational modification (PTM) involved in many important physiological processes such as macrophage polarization, immune regulation, and tumor cell growth. However, traditional methodologies for studying lactylation have predominantly relied on peptide enrichment from whole-cell lysates, which tend to favor the detection of high-abundance peptides, thus limiting the identification of low-abundance lactylated peptides. To address this limitation, here, we employed subcellular fractionation to separate proteins and map lactylated peptides from each isolated subcellular fraction using a model cell line. In brief, we identified 1,217 lysine lactylation (Kla) sites on 553 proteins across four subcellular fractions. Subsequent pathway enrichment analysis revealed that Kla proteins participate in distinct pathways depending on the subcellular contexts. In addition, this subcellular fractionation method enabled the discovery of 36 previously unreported Kla proteins and 223 novel Kla sites, many of which are present in low abundance. Notably, several proteins contain multiple newly identified Kla sites, exemplified by the transcriptional regulator ATRX. Furthermore, our results indicate the possibility of PTM crosstalk between Kla and other PTMs such as ubiquitination and sumoylation. In conclusion, subcellular fractionation facilitates the identification of Kla proteins that have been previously uncovered and could be overlooked by affinity enrichment of whole-cell lysates.