Journal of the American Society for Mass Spectrometry最新文献

Native mass spectrometry (MS), ion mobility (IM), and collision-induced unfolding (CIU) have all been widely used to study the binding of small molecules to proteins and their complexes. Despite many successes in detecting subtle gas-phase stability differences in smaller systems dominated by single-domain subunits, studies targeting complexes comprised of large, multidomain subunits still face many challenges. For example, polyketide synthases (PKSs) are multiprotein enzymes that use their modular architecture to produce polyketide natural products and form the basis for nearly one-third of pharmaceuticals. Here, we describe the development of CIU methods capable of extracting information from these multiprotein complexes and demonstrate the current limits of quantitative CIU technology by probing the stabilities ∼280 kDa PKS dimer protein complexes. Our approach detects the evidence of the stability shifts associated with substrate binding that accounts for <0.1% of the mass for the intact assembly.

Phased structures for lossless ion manipulation offer significant improvements over the scanning second gate method for coupling with ion trap mass analyzers. With an experimental run time of under 1 min for select conditions and an average run time of less than 4 min, this approach significantly reduces experimental time while enhancing the temporal duty cycle. The outlined SLIM system connects to an ion trap mass analyzer via a PCB stacked ring ion guide, which replaces the commercial ion optics and capillary inlet. By applying a discrete and repeating injection pulse and solving a series of algebraic equations, the system reconstructs an arrival time distribution with a minimal degree of error with enhanced ion throughput. To demonstrate the feasibility of this approach, the 3.4-m SLIM system resolves gas-phase conformers for various small peptides and proteins. This system and methodology also enable direct implementation between SLIM and ion trap mass analyzers traditionally interfaced with front separation systems such as liquid chromatography.

Coenzyme Q₁₀ (CoQ₁₀) and closely related compounds with varying isoprenoid tail lengths (CoQ_n, n = 6-9) are biochemical cofactors involved in many physiological processes, playing important roles in cellular respiration and energy production. Liquid chromatography (LC) coupled with single or tandem mass spectrometry (MS) using electrospray (ESI) or atmospheric pressure chemical ionization (APCI) is considered the gold standard for the identification and quantification of CoQ₁₀ in food and biological samples. However, the characteristic fragmentation exhibited by the CoQ₁₀ radical anion ([M]^•-, m/z 862.684), the prevailing ion generated by APCI in negative polarity, has not been studied in detail. In this work, a systematic study was carried out to clarify this issue, using higher collisional energy dissociation (HCD) with high-resolution tandem FTMS and collision-induced dissociation-low-resolution sequential mass spectrometry (CID-MSⁿ, n = 2-4). Various fragmentation pathways were successfully interpreted, with some structures proposed for product ions checked using density functional theory (DFT) calculations. Besides the already-known detachments of methyl radicals occurring directly from the CoQ₁₀ radical anion and leading to ions like [M - CH₃]^- and [M - 2CH₃]^•-, the homolytic cleavage of C-C bonds along the oligo-isoprenoid side chain was tentatively proposed to explain some of the observed fragmentations. As a result, the generation of uncommon yet potentially stable distonic biradical anions was hypothesized, with some of them likely undergoing intramolecular cyclization to generate ions without unpaired electrons. Diagnostic product ions emerged from the fragmentation processes of CoQ₁₀ and were found to be common also to the radical anions of other CoQ_n derivatives (n = 7-9), facilitating their identification in extracts of edible Brassicaceae plant microgreens by reversed-phase liquid chromatography (RPLC)-APCI-FTMS.

In this communication we report the construction of a printed circuit board which mounts directly to the vacuum chamber of a mass spectrometer and produces the RF waveforms needed by many nonmass-selective devices such as ion guides and ion funnels. Our device is designed to replace a standard KF40 flange, can maintain vacuum chamber pressures of less than 10^-6 Torr, and contains the circuitry of the open-source Wisconsin Oscillator RF power supply to generate RF waveforms of 1-4 MHz and up to 200 V_p-p. In this iteration of the Wisconsin Oscillator, we also introduce a variable resistor to control the output RF amplitude and show that its ion transmission capabilities are identical to those provided by commercial RF power supplies. With this new implementation we have greatly reduced the space and monetary requirements for driving nonmass-selective ion manipulation devices, which we expect to be advantageous to those developing low-cost and/or portable mass spectrometry systems.

Atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) is a widely used technique for the analysis of a diverse range of analytes. Under APCI conditions, a nonthermal plasma, rich in highly oxidative species such as H₂O₂, O₃, atomic O, and radicals such as HO^•, is created. These oxidants trigger unanticipated and often undesirable chemical reactions within the ion source. For example, when aniline was introduced into this environment, it initially underwent oxidative dimerization forming hydrazobenzene (m/z 185). However, with prolonged exposure, there was a marked increase in total ion abundance and the generation of additional artifact ions such as protonated azobenzene (m/z 183) and protonated azoxybenzene (m/z 199). The emergence of these artifacts was found to be highly dependent on the corona-current magnitude. Moreover, the desorption-gas temperature significantly influenced the rate of artifact generation. Recognizing and acknowledging the formation and presence of such artifacts in an ion source is paramount in conducting validated chemical analysis. The existence of artifacts can complicate mass spectral interpretation, potentially leading to erroneous conclusions and misinterpretations of both qualitative and quantitative data. Thus, understanding the intricacies of nonthermal plasma-driven artifact formation is critical for accurate analytical outcomes.

Substances of misuse are becoming increasingly difficult to analyze as unique methods of smuggling are adopted and due to the rapid emergence of new psychoactive substances, increasing the pool of compounds to characterize and identify. Technologies such as gas chromatography and liquid chromatography coupled to mass spectrometry (MS) represent the gold standard for accurate and robust analysis, with on-site ambient- and portable-MS systems providing rapid methods of drug screening and testing. For many samples containing residual analyte quantities, methods to improve sensitivity through chemical derivatization are critical for accurate determination. Herein, we demonstrate for the first time the use of trimethylation enhancement using diazomethane (TrEnDi) to improve the MS-based sensitivity of 13 different drugs of misuse. All analytes were successfully permethylated, with 11 demonstrating improved analytical characteristics from TrEnDi with MS sensitivity enhancements ranging from 1.2-fold to as high as 24.2-fold in the case of psilocybin, as well as increases in reversed-phase chromatographic retention for most species. Derivatization using ¹³C-isotopically labeled TrEnDi reagents were used to successfully resolve isobaric interference issues between three pairs of controlled substances. By using an unconventional aprotic solvent system for electrospray ionization, the benefit of a fixed-permanent positive charge was highlighted as TrEnDi-modified amphetamine was easily measured while unmodified was not detected. Finally, TrEnDi was employed to boost the sensitivity of morphine in a real urine matrix. Our results demonstrate a percent recovery of 103.1% and a sensitivity enhancement of 2.4-fold, demonstrating the versatility and applicability of TrEnDi to pre-existing analytical workflows for trace analysis.

Illicit fentanyl and fentanyl analogs are a growing concern in the United States as opioid related deaths rise. Given that fentanyl analogs are readily obtained by modifying the structure of fentanyl, illicit fentanyl analogs appearing on the black market often contain similar structures, making analogue differentiation and identification difficult. Thus, obtaining both precursor and product ion data during analysis is becoming increasingly valuable in fentanyl analog characterization. In this paper, we provide GC column retention time, precursor, and product ion mass spectrum data for 74 fentanyl analogs that were analyzed using atmospheric pressure chemical ionization-gas chromatography–mass spectrometry (APCI-GC-MS) utilizing a triple quadrupole mass analyzer. During analysis, precursor ions underwent collision induced dissociation (CID) by increasing the collision energy (10, 20, 30, 40, and 50 V) throughout a single run. Data reveal that APCI readily produces product ions of the piperidine and N-alkyl chain but rarely provides data on the acyl group. Furthermore, fentanyl analogs with greater substitution at the N-alkyl chain demonstrate a greater preference for dissociation at the N-αC and αC-βC bond, while greater substitution at the amide group leads to fragmentation at the N–C4 bond.

Deamidation of asparagine and glutamine residues occurs spontaneously, is influenced by pH, temperature, and incubation time, and can be accelerated by adjacent amino acid residues. Incubation conditions used for proteolytic digestion in bottom-up proteomic studies can induce significant deamidation that affects results, either knowingly or unknowingly. This has prompted studies into modifications to common trypsin digestion protocols to minimize chemical deamidation, including shorter incubation times and specific lysis buffers. Prior work suggested ammonium acetate at pH 6 to minimize chemical deamidation, but this buffer has compatibility issues with trypsin digestion and common assays (e.g., bicinchoninic acid assays). Here, we re-evaluated former comparisons of Tris-HCl, ammonium bicarbonate, and triethylammonium bicarbonate buffers for the amount of artificial, chemically induced deamidation generated in a standard bottom-up proteomics workflow, and we added an evaluation of three commonly used and biologically compatible buffers, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), EPPS (3-[4-(2-Hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid), and PBS (phosphate buffered saline). Our findings show that HEPES exhibited the least amount of artificial deamidation and is a reasonable choice for general proteomic experiments, especially for studies considering N-glycosylation.