Journal of the American Society for Mass Spectrometry最新文献

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.

Mass spectrometry imaging (MSI) is a powerful technique for studying the spatial localization of N-linked glycans in biological tissues, which are important biomarkers for various diseases. Analyzing N-linked glycans requires the deposition of an enzyme onto a biological tissue section to release them from proteins. However, existing equipment for enzyme deposition is relatively expensive and may not be readily accessible to some laboratories. To address this challenge, we developed and evaluated a cost-effective approach for enzyme application onto tissue sections using a mini-humidifier. We demonstrate the capabilities of this approach by applying peptide N-glycosidase F (PNGase F) for MSI of N-linked glycans present within mouse brain tissue sections using nanospray desorption electrospray ionization (nano-DESI). The performance of the mini-humidifier was comparable to that of a widely used HTX-TM Sprayer, establishing it as a cost-effective and efficient alternative for enzyme deposition onto tissue samples in MSI experiments. This work offers an accessible approach for enzyme application on biological samples to study the spatial distribution of N-linked glycans using MSI.

Noncovalent interactions play an important role in the way protein structures and protein-protein interactions are stabilized. Mapping these interactions at a molecular level is crucial as peptide complexes serve as models for protein-protein interfaces. In this work, complementary analyses using trapped ion mobility spectrometry (TIMS), tandem ECD/UVPD MS fragmentation, and molecular dynamics were applied to the study of native peptide-peptide complexes. In particular, three complexes representing peptide-peptide intramolecular interactions of the intrinsically disordered high-mobility group AT-Hook2 (HMGA2) protein were described. AT-hook peptides, when complexed with the C-terminal tail (CTMP), showed a higher gas-phase stability for ATHP1-CTMP, followed by ATHP2-CTMP and ATHP3-CTMP. High sequence coverage was obtained by using ECD and UVPD fragmentation for the single peptides (∼100%) and the peptide-peptide complexes (∼75%). At least three peptide-peptide structures were separated in the mobility domain for the ATHP-CTMP complexes. All three complexes showed high structural diversity and the possibility of being aligned in forward and backward orientations.

Reliable mass accuracy is essential for the confident identification of diagnostic fragment ions in high-resolution mass spectrometry. During the analysis of perfluoroalkyl sulfonic acids (PFSAs) with a Q Exactive Orbitrap, we consistently observed the characteristic O₃Ṡ^- fragment, expected at a mass-to-charge ratio (m/z) of 79.9574, reported at m/z 79.9568, a systematic deviation of approximately -6 parts per million (ppm). Similar errors affected other ions below m/z 100, while precursors and higher-m/z fragments remained within the specification. Comparison with an Exploris 120 instrument, calibrated with anchors down to m/z 59, confirmed that the deviation is not intrinsic to the ion but originates from the limited calibration range of the Q Exactive standard calibration solution. We demonstrate that extending calibration to include additional low-m/z anchors generated in situ by in-source fragmentation fully corrects the error without affecting the accuracy at a higher m/z. This adjustment resolves systematic deviations for ions below m/z 100 in the Q Exactive instruments.

Stable and long-lived radioactive isotopes are ubiquitous in nature and serve as unique tracers across diverse fields such as nuclear astrophysics, atmosphere chemistry, hydrology, environmental chemistry, and the diagnosis of diseases in the human body. Over the past decades, accelerator mass spectrometry and spectroscopic methods have been used to measure the abundance of stable and long-lived radioactive isotopes. However, their accuracy has been constrained by the systematic uncertainties inherent in sophisticated instrumentation and limitations in the abundance sensitivity. Here, we present a novel approach based on the fundamental mechanism of molecular Coulomb explosion fragmentation (i.e., molecules breakup as a result of Coulomb repulsion between the positively charged nuclei within molecules that are rapidly stripped of their electrons), utilizing a two-dimensional coincidence time-of-flight spectrometer to detect fragmented isotopic ion pairs. The present method enables direct determination of the isotopic abundances of ¹³C and ¹⁸O with an accuracy better than 0.02%, significantly improving abundance sensitivity by powerful identification and eliminating systematic uncertainties. Our molecular Coulomb explosion spectrometry provides high-accuracy measurement of stable and long-lived radioactive isotope abundance, with significant potential to advance isotope tracer studies in the Earth environment, anthropology, archeology, global ecological cycles, fundamental nuclear physics, and biomedicine.