Journal of the American Society for Mass Spectrometry最新文献

Microdroplet digestion of proteins, including monoclonal antibodies (mAbs), has gained increasing attention for its potential in rapid protein structural characterization. In this study, we developed a fast antibody characterization and quantitation method using online microdroplet trypsin digestion. This automated approach involves ultrafast digestion in <1 ms (>90% digestion efficiency) and subsequent MS and MS/MS for peptide mapping and pinpointing peptide modifications. For instance, by MS/MS analysis, the N³⁸⁷ residue rather than N³⁹² and N³⁹³ of the "PENNYK" peptide, GFYPSDIAVEWESN³⁸⁷GQPEN³⁹²N³⁹³YK generated from NIST mAb microdroplet digestion, was found to undergo major deamidation due to its proximity to the G³⁸⁸ residue (upon incubation with a pH 8 Tris buffer for 5 days, ca. 32% of the N³⁸⁷ residue was deamidated). To further demonstrate our method's applications, methionine oxidation, another important type of post-translational modification (PTM) of mAbs, was successfully quantified by spiking the mAb sample with standard peptides for microdroplet digestion. Such an absolute quantitation approach showed a better accuracy for measuring the methionine oxidation level, in comparison to traditional relative quantitation methods, which simply compare ion intensities of oxidized and intact peptides to calculate the oxidation level (the relative quantitation is problematic as the oxidized and intact peptides have different ionization efficiencies). Similarly, by spiking the antibody with a heavy isotope-labeled antibody, our microdroplet digestion method allowed quick absolute antibody quantification, demonstrating good linearity (R² = 0.99), sensitivity (LOD: 1.2 ng), and accuracy (0.6-10% quantification error in comparison to the theoretical amount of injected antibody).

The practical application of advanced oxidation processes is often constrained by their high energy consumption and dependence on chemical additives. To overcome these limitations, this work harnesses the highly reactive species water dimer radical cation (H₂O)₂^+•, as a green oxidant for efficient dye degradation. A corona discharge setup was developed for the controlled, ambient generation of (H₂O)₂^+•, which was directly detected by in situ mass spectrometry. Electron paramagnetic resonance spectroscopy further verified its role as a precursor to hydroxyl radicals, elucidating the underlying radical cascade. Using model dyes (e.g., rhodamine B) as representative pollutants, degradation was quantitatively tracked via their characteristic UV–vis absorption. The (H₂O)₂^+•-based process achieved 99% removal of rhodamine B in 8 min without any external chemical inputs, representing a more than 1100-fold enhancement in processing rate over conventional Fenton oxidation. Scalability was demonstrated through an enlarged array device, which exhibited an energy consumption of 0.75 kJ/mg and a degradation efficiency ∼90 times higher than that of a standard Fenton system. This study establishes a practical route to utilize (H₂O)₂^+• for rapid, chemical-free water treatment, offering a scalable and energy-efficient strategy for green degradation technologies.

Sc237 and 139H are two phenotypically different prion strains of hamster (Mesocricetus auratus) adapted scrapie. Each replicates by inducing the natively expressed hamster PrP^C to adopt its infectious conformation. The nine methionines in hamster PrP^C can be oxidized to the corresponding methionine sulfoxide by hydrogen peroxide. The extent of this oxidation is determined by the methionine's conformation-dependent surface exposure. Methionine sulfoxides are unaffected by protein denaturation. Samples of 139H and Sc237 prions were untreated or digested with proteinase K (PK), isolated by ultracentrifugation, oxidized in 0 mM or 50 mM hydrogen peroxide, inactivated by denaturation, reduced/alkylated, and then digested with trypsin, trypsin/chymotrypsin, or Arg-C. Seven peptides, TNMK, HMAGAAAAGAVVGGLGGY, MLGSAMSR, PMMHFGNDWEDR, ENMNR, IMER, and VVEQMCTTQYQK, resulted from the enzymatic digestion. These peptides contain the nine methionines in hamster PrP and were analyzed using an MRM-based approach to determine the extent of each methionine's oxidation. Differences in the extent of methionine oxidation were observed for each strain. These differences were observed in PK digested and untreated samples. Hamster and sheep PrP share six methionines and comparison of the methionine oxidation in Sc237, 139H, and sheep scrapie showed different methionine oxidation patterns. This approach is a form of conformational sequencing that can be used to compare the surfaces of prion strains from the same species and prions from different species.

Photon activation is a powerful means of inducing controlled fragmentation in mass spectrometry, but the simultaneous activation of multiple precursor ions may produce multiplexed tandem mass spectra that are difficult to interpret. In this work, we show that such photon-activated multiple precursor spectra can be decomposed into individual precursor contributions using correlation-based and information-theoretic approaches. We propose a general, data-driven framework for the statistical resolution of multiplexed photon-activated MS/MS spectra based on the statistical treatment of the spectra followed by clustering analysis. This approach allows statistical tandem mass spectra to be obtained without prior knowledge of the precursor identities.

This study examines the decomposition of the protonated dipeptide, AsnSer methylated on the serine side chain, Asn(OMe)Ser. We utilize threshold collision-induced dissociation (TCID) conducted on a guided ion beam tandem mass spectrometer (GIBMS) to examine the deamidation and dehydration from [Asn(OMe)Ser+H]⁺. We also use infrared multiple-photon dissociation (IRMPD) spectroscopy to verify the reactant and product structures. These experimental analyses are reported in parallel with complementary quantum-chemical calculations, where key reaction energies are determined at the B3LYP, ωB97XD, and MP2(full) levels of theory. Comparison of IRMPD and theoretical spectra identifies the major deamidation product as a furanone with a probable contribution of a succinimide. Dehydration occurs through the formation of multiple products, but we assign the primary product to be a diketopiperazine along with a minor contribution from a pyrrolidone. The TCID data were modeled and the results show that deamidation begins at 141 ± 7 kJ/mol and dehydration at 121 ± 7 kJ/mol. Compared to the unmethylated analogue, the deamidation threshold is comparable whereas that for dehydration is elevated, consistent with methylation shutting down the lowest-energy pathway for dehydration of [AsnSer+H]⁺, previously shown to form an oxazoline product ion.

In metabolomics, tandem MS (MS²) fragmentation libraries are important for the identification of unknown features, but generating these libraries takes many valuable hours of instrument and operator time. Here, an immediate droplet-on-demand/open port sampling interface was used to rapidly acquire tandem MS of standards arrayed in a 96-well plate format. A workflow was developed for automated, high-throughput control of MS² library generation. Pure standard mass spectral libraries were collected on Orbitrap and Q-TOF mass spectrometers for 192 compounds using 6 different collision energies with a throughput of 4 and 7.8 s/spectrum, respectively. Libraries were acquired using different solvent additives, precursor adducts, and ion polarities.

Cellular functions arise from the coordinated action of proteoforms, which typically form multiproteoform complexes (MPCs), rather than functioning as isolated molecular entities. Deciphering the architecture and composition of MPCs is essential for linking proteoform diversity to biological function. Native top-down (nTD MS) and complex-down mass spectrometry (CxD MS) have emerged as powerful strategies to characterize MPCs, offering intact mass analysis as well as gas-phase sequencing either at the level of the complete assembly or its constituent proteoform subunits. Because the attainable sequence coverage is highly influenced by the ion activation technique, expanding activation strategies is key to improving proteoform characterization. To this end, we implemented infrared (IR) activation for the analysis of soluble MPCs─alcohol dehydrogenase (ADH; 147 kDa tetramer), enolase (96 kDa dimer), and pyruvate kinase (PK; 232 kDa tetramer). IR photons were used to induce infrared multiphoton dissociation (IRMPD) and to enhance electron-based fragmentation via activated-ion electron transfer dissociation (AI-ETD), and performance was benchmarked against higher-energy collisional dissociation (HCD). For ADH (∼36 kDa subunits), AI-ETD, HCD, and IRMPD returned similar sequence coverages in nTD MS experiments (36, 38, and 34%, respectively), with complementary cleavages resulting in a combined 48% coverage. As subunit mass increased, radical-driven fragmentation provided a clear advantage: for PK (∼57 kDa subunits), AI-ETD achieved 28% sequence coverage─approximately 15% higher than HCD or IRMPD. Together, these results highlight IR irradiation─both as a standalone dissociation modality and as a complement to electron-based activation─as a versatile strategy to enhance proteoform-level sequencing in native and complex-down MS workflows.

The widespread presence of pharmaceuticals, including antibiotics, in our aquatic environment raises important societal concerns. When studying their environmental fate, stable isotope analysis of nitrogen and carbon at natural abundance offers unique insight into source fingerprinting and degradation-associated kinetic isotope effects. Here, we synthesized compound-specific reference standards to enable electrospray ionization (ESI) Orbitrap mass spectrometry (MS) for fragment-specific carbon and nitrogen isotope analysis (Δδ¹³C and Δδ¹⁵N) of sulfamethoxazole (SMX), a most frequently detected antibiotic. Fragment-specific isotope analysis relied on fragmentation of SMX ions in the collision cell, resulting in two fragment ions representing the aniline part (m/z = 92, F92) and the 3-amino-5-methylisoxazole ring (m/z = 99, F99) of SMX. Reference materials were prepared (i) through total synthesis of SMX from labeled precursors that resulted in specific positions labeled with ¹³C and ¹⁵N, (ii) followed by the mixing of labeled SMX with SMX at natural abundance. The bulk isotope values of these in-house standards were determined by elemental analysis isotope ratio mass spectrometry and used for calibration of the ESI-Orbitrap-MS method. Injecting standards directly into the ESI-Orbitrap-MS resulted in 95% confidence intervals (CIs) of 0.7‰ and 3.4‰ for Δδ¹³C and Δδ¹⁵N in F92, respectively, and 1.3‰ and 2.9‰ for Δδ¹³C and Δδ¹⁵N in F99, for quintuplicate measurements of standards. A proof-of-principle demonstration shows that this approach could indeed successfully quantify changes in fragment-specific isotopic signatures, Δδ¹³C and Δδ¹⁵N, during degradation of SMX.

Nanoflow electrospray ionization is commonly used for proteomics due to its high sensitivity. Signal intensity, however, is dependent on optimal emitter positioning relative to the mass spectrometer inlet. Here, we characterize the effect of varied emitter positions on peptide signal intensity in all three dimensions using emitters and flows consistent with standard proteomic analyses. We observe improved signal robustness to x/y variations at increasing z distances and demonstrate that positioning within 1 to 2 mm of the optimal location will maintain consistent signal. Signal intensity behavior is consistent across the m/z range, suggesting emitter positions do not need to be fine-tuned for different analytes for proteomics analyses. These results provide insight for proteomics researchers using nanoflow LC–MS/MS.

Mass spectrometry (MS)-based protein footprinting and, more specifically, fast photochemical oxidation of proteins (FPOP) are methods that have been found to be important for studying proteins, their structures, and their relationships to other proteins or ligands. In-cell FPOP (IC-FPOP) was developed to study proteins in their native environment. Initial work with IC-FPOP has been performed using a platform incubator with an XY movable stage (PIXY). However, low throughput and a six-well plate format restricted the experiment by limiting the number of technical replicates that can be analyzed at one time and requiring large amounts of samples per experiment. Here, we introduce an improved, higher throughput platform that allows IC-FPOP to be run on a fully automated XY stage (AXYS) using 24-well plates. Comparison with the PIXY system results shows that this platform can successfully modify more proteins in less time. AXYS also increases the types of biological samples that can be analyzed by IC-FPOP.