Journal of the American Society for Mass Spectrometry最新文献

Machine learning (ML) accelerates progress in many areas, including biomedical and clinical research. ML algorithms provide powerful options for efficiently analyzing multivariate data sets. We developed and validated an ML pipeline to detect myelodysplastic syndrome (MDS)-associated pathological alterations of extracellular matrices (ECMs) by time-of-flight secondary ion mass spectrometry (ToF-SIMS). A Bayesian-optimized neural network (NN) was trained and applied to classify ToF-SIMS spectra of ECM secreted by mesenchymal stromal cells (MSCs) derived from MDS patients and healthy reference donors. Validated by principal component analysis, the explainer tool SHapley Additive exPlanations (known as SHAP) was integrated into the analysis pipeline to unravel characteristic compositional and structural differences of the ECM variants. Our results demonstrate the potential of ToF-SIMS-ML for the label-free investigation of pathogenic alterations of the ECM. Integrated into the multiscale ECM analysis of cell and organoid-based disease models, the introduced methodology may facilitate advances in the development of novel diagnostic and therapeutic strategies.

Molecules that possess more than one protonation site can form protonation site isomers (protomers) in mass spectrometry. During the ionization process, a change in protonation site can alter the resulting collision-induced dissociation mass spectrum, which can hamper the accurate confirmation of unknown analytes in mass spectrometry workflows. In this study, we report the presence of protomers for ten fluoroquinolone antibiotics in a conventional matrix-assisted laser desorption ionization (MALDI) ion source and compare this against an atmospheric pressure MALDI plasma postionization (AP-MALDI-PPI) source. Irrespective of matrix composition, only the most stable protomer forms in a conventional MALDI ion source, which is consistent with a thermodynamically driven MALDI ionization process. In contrast, between 1-3 protomers form with an AP-MALDI-PPI source and this demonstrates that a different mechanism is responsible for analyte protonation. Protomer populations can be biased to favor the most stable protomer with high proton affinity MALDI matrices. Protomer populations can also be biased by doping the plasma with methanol or acetonitrile solvent vapor. Fluoroquinolone protomers are separated by trapped-ion mobility spectrometry (TIMS) and subjected to collision-induced dissociation. The protonation sites are assigned by the presence of unique fragment ions and comparing experimentally derived collisional cross sections ^TIMSCCS_N2 against trajectory method CCS_N2 calculations at the ωB97X-D/aug-cc-pVDZ//ωB97X-D/cc-pVDZ level of theory. This composite study shows that protomer population ratios change between MALDI and AP-MALDI-PPI ion sources, sample preparation method, and analyte structure, which can affect their ion mobility distributions and potentially impact their faithful confirmation.

Native mass spectrometry (nMS) has emerged as a complementary approach for elucidating molecular parameters of biological complexes relative to solution-phase experiments. Herein, we utilize nMS to determine the subunit binding affinities (K_d,i) of the single-stranded DNA binding protein (SSB) from Saccharolobus solfataricus (Sso) to poly dT single-stranded DNA (ssDNA) compared with the apparent K_d' values obtained from solution-phase fluorescence anisotropy. This work resolves conflicting previous biochemical reports on the stoichiometry and affinities of SsoSSB while also highlighting the advantages and limitations of nMS quantification. Covalent concatemers of SsoSSB with increasing molecular weights were utilized as response factor (RF) standards to correct for physical and instrumental parameters that systematically underrepresent abundances from ionization of larger mass species. Furthermore, we show that regardless of the nMS quantification metric (peak area or intensity), meaningful comparative data can be extracted from multicomponent biochemical systems. Importantly, the binding affinities of the individual species (K_d,i) determined by nMS approach the apparent K_d' from bulk solution-phase measurements but have the added benefit of separately quantifying individual binding steps within a multistep assembly process. Interestingly, the stoichiometries of SsoSSB binding to 15 or 30 nucleotides of ssDNA measured by nMS are subsaturating. The calculated binding affinities of the first and second SsoSSB molecules show some positive cooperativity, while the binding of the third is an order of magnitude weaker, suggesting that negative cooperativity is utilized to limit binding near the ends of the available length of the ssDNA.

Reduction of disulfide bonds in proteins followed by selective modification of Cysteine (Cys) residues is a common practice in proteomics experiments to facilitate digestion into peptides and ultimately identification by mass spectrometry (MS). Due to its high nucleophilicity, a variety of reaction pathways can be used to block Cys; however, arylation is not often employed. Recently, Lipka and co-workers reported an electrophilic Cys arylation reagent, N-methyl-2-methylsulfonylpyridinium (CAP4), that can arylate free thiols with high selectivity and rate (Lipka, B. M.; Honeycutt, D. S.; Bassett, G. M.; Kowal, T. N.; Adamczyk, M.; Cartnick, Z. C.; Betti, V. M.; Goldberg, J. M.; Wang, F. Ultra-Rapid Electrophilic Cysteine Arylation. J. Am. Chem. Soc. 2023, 145 (43), 23,427-23,432). Additionally, CAP4 modification includes the addition of a fixed charge to the side chain that could potentially influence MS experiments. The fixed charge could be useful if it affords better sensitivity or detrimental if it negatively affects fragmentation. Herein, we show that CAP4 modification leads to favored fragmentation pathways involving the modified side chain in both higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD). Abundant side chain losses from CAP4 are observed in HCD, while in ETD, electron transfer leads to the formation of a hydrogen deficient beta radical at the Cys residue. Formation of the beta radical is shown to be charge-state dependent, and collisional activation of the radical leads to radical-directed dissociation (RDD)-derived fragments. In addition, we evaluated the potential of CAP4 in a bottom-up proteomics workflow using a model protein mixture. Despite its influence on fragmentation, rapid arylation (5 min) by CAP4 resulted in comparable sequence coverage and total modified Cys-containing peptide IDs when compared to 1 h iodoacetamide modification.

High-throughput analysis is becoming increasingly important in liquid chromatography-mass spectrometry coupling to minimize downtime for efficient and cost-effective operations in the analytical laboratory. In response to this need, a dual ion source has been designed to provide two ionization modes under atmospheric conditions: electrospray ionization (ESI) and dielectric-barrier discharge (DBD)-based plasma. This dual ion source allows for the rapid switching of the ionization mode during a chromatographic run. Therefore, substances with different ionization behaviors can be detected within one measurement if their retention times differ. The performance of the dual ion source was evaluated using single- and multidimensional liquid chromatography coupled to a quadrupole-time-of-flight (Q-TOF) mass spectrometer and a structures for lossless ion manipulations ion mobility mass spectrometer to analyze mixed standards and real samples. Most notably, the dual ion source maintained strong ion signals and a stable performance while switching ionization modes during a run. Instrumental limits of detection reached down to the sub 1 nM range for both ESI and DBD. This work describes the design and construction of the dual ion source, which provides electrospray- and plasma-based ionization that can be switched within a chromatographic run.

Top-down proteomics is primarily performed using electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in the positive mode. Development of methods in the negative mode can potentially facilitate analysis of acidic proteome, but has been hampered by the low ionization efficiency and the lack of effective fragmentation methods for protein anions. Here, we investigate the performance of ultraviolet photodissociation (UVPD) for top-down analysis of protein anions. We employed organic bases as additives in solution to yield highly charged, nonadducted protein anions of high abundance. We compared UVPD with higher energy collisional dissociation (HCD) and activated electron photodetachment (a-EPD) for fragmentation of proteins ranging from 8.6 to 47 kDa. UVPD yielded abundant charge-reduced precursor radicals, in addition to numerous a/x, b/y and c/z fragment ions. UVPD offered 70-95% sequence coverage for proteins below 20 kDa regardless of the presence of disulfide bonds, and 30% coverage for the largest protein studied (47 kDa enolase), higher coverage than HCD and a-EPD. UVPD of deprotonated proteins exhibited several features similar to those of protonated proteins, such as minimal sensitivity to the charge state, production of abundant a/x fragment ions, and fairly uniform backbone cleavages adjacent to each residue (i.e., no prominent preferential cleavage sites).

Effective glycopeptide identification with tandem mass spectrometry (MS/MS) often relies on both low mass-to-charge (m/z) ions derived from glycan-specific oxonium ions and higher m/z peptide fragment ions that retain glycan modifications. Thus, glycoproteomic experiments benefit from a wider MS/MS scan range, i.e., the breadth of m/z values measured in fragmentation spectra, than those typically used in nonmodified peptide analyses. Here, we explore the implications of breaking a common axiom for scan range settings called the "5-10-15 rule" used to maximize transmission of ion populations of interest. This 5-10-15 rule, which defines the upper m/z value for a scan as a multiple of the first m/z value, comes from fundamental requirements for stable ion trajectories, where voltage settings must balance retention of low m/z ions while also generating effective pseudopotential wells to trap high m/z ions. Adhering to this calculation for MS/MS scan range settings can reduce glycopeptide ion coverage by excluding the analysis of either low m/z oxonium ions or high m/z fragment ions. We use a quadrupole-Orbitrap-linear ion trap Tribrid MS system (Orbitrap Ascend) to investigate the implications of following or breaking the 5-10-15 rule in MS/MS scans for glycopeptide characterization with higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer/higher-energy collision dissociation (EThcD). For scans with a first m/z value around m/z 120 (i.e., capturing most common glycan-specific oxonium ions), we show that breaking the 5-10-15 rule does not lead to a significant loss of fragment ion transmission at either extreme of the m/z range. We use this case study to discuss the concepts important to using the 5-10-15 rule wisely and when it can be practically ignored, such as using large scan ranges to improve glycopeptide characterization.

Effective differentiation of chiral metabolites, coupled with precise determination of their enantiomeric ratios, is crucial for advancing biomarker screening and gaining comprehensive insight into the etiology of metabolic disorders. Herein, we integrate a chiral derivatization strategy with matrix-assisted laser desorption/ionization (MALDI) coupled with trapped ion mobility spectrometry (TIMS) for chiral discrimination of α-hydroxy acids, 2-hydroxyglutarate (2-HG) and lactic acid (LA), in complex matrices. Systematic optimization of derivatization reagents N-(p-Toluenesulfonyl)-l-phenylalanyl chloride (TSPC) and diacetyl-l-tartaric anhydride (DATAN), with alkali metal ion adducts, enables high-resolution enantiomer separation of 2-HG and LA via diastereomeric conversion. This method exhibits an exceptional ability to determine enantiomeric ratios as low as 5.8%. The utility of this method is presented for sensitive identification of 2-HG in isocitrate dehydrogenase (IDH)-mutated tumor tissues and the precise determination of LA enantiomers in fermented foods, thus underscoring the significant potential for advancing research in food safety, pharmaceutical development, and clinical diagnostics.

Laser-induced acoustic desorption (LIAD) enables the soft volatilization of nonvolatile and thermally labile compounds as neutral molecules from metal surfaces. Concurrently, doping with dichloromethane (CH₂Cl₂) during vacuum ultraviolet photoionization (PI) has been established as a highly efficient protonation method for oxygenated volatile organic compounds. In this study, an experimental apparatus based on a krypton VUV lamp, a 355 nm pulsed laser, and a time-of-flight mass spectrometer was set up to investigate LIAD/PI and LIAD/dichloromethane-enhanced vacuum ultraviolet photoionization (abbreviated as EPI) mass spectra of five representative amino acids─valine (Val), serine (Ser), aspartic acid (Asp), histidine (His), and glycine (Gly). The obtained LIAD/PI and LIAD/EPI mass spectra revealed that the main ion species of both LIAD/PI and LIAD/EPI for the five amino acids were the protonated molecular ions ([M + H]⁺) and the fragment ions resulting from the combined loss of H₂O and CO (denoted as [M_d + H]⁺). The fragment-to-parent ion ratios of the LIAD/PI for Val, Ser, Asp, His, and Gly were 0.95, 0.45, 0.29, 0.71, and 0.03, while the corresponding ratios of LIAD/EPI were 0.63, 0.26, 0.21, 1.19, and 0.05, confirming that LIAD/EPI is a soft ionization technique comparable to LIAD/PI. In contrast to the LIAD/PI process, the LIAD/EPI signal intensities of the amino acids were remarkably enhanced via doping with dichloromethane. The best observed enhancing factors of the signal intensities (I_EPI ([M + H]⁺)/I_PI ([M + H]⁺)) were 637, 559, 354, 80, and 716 times for Val, Ser, Asp, His, and Gly, respectively, derived from the LIAD/PI and LIAD/EPI mass spectra of the five amino acids mixture. In addition, the protonated dimers of the amino acids ([2M + H]⁺) were observed in the LIAD/PI and LIAD/EPI processes, along with their fragment ions resulting from the loss of CO, H₂O, and NH₃ (denoted as [(2M)_d + H]⁺). The key experimental parameters, including the carrier gas flow rate, the doped CH₂Cl₂ concentration, and the LIAD laser intensity, were investigated and reported in the paper. This study demonstrates that the LIAD/EPI method is a significantly more efficient ionization method for amino acids compared with the conventional PI method.