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Detection of illicit compounds like explosives and drugs of abuse at trace levels is crucial to provide public security and health safety. A dual ambient sampling system hollow cathode discharge (HCD) ion source was developed to investigate its performance. Here, trinitrotoluene (TNT), trinitrobenzene (TNB), hexamethylene triperoxide diamine (HMTD), and triacetone triperoxide (TATP) as explosives and methamphetamine (MA) as drugs of abuse were taken as model compounds. Two sample inlets, inlet-1 and inlet-2, are available for ambient sampling. In negative ion mode, N2 and air HCD plasmas are confined close to inlet-1, but in positive ion mode, they are confined close to inlet-2. Special design of the ion source makes it feasible to generate multiple ions from a single analyte, which assists in understanding the gas phase ionization mechanism. In negative ion mode, both TNT and TNB gave radical ions, [M]−•, as major ions for N2 HCD plasma as they were introduced via inlet-1 or inlet-2. TNB gave radical ions for air and N2 HCD plasmas, while TNT exhibited adduct ions, [TNT–H]−, by using air HCD plasma. In positive ion mode, HMTD gave [HMTD + H]+m/z 209 ions, while TATP only produced adduct ions with ammonia, [TATP + NH4]+m/z 240. Regardless of ion source inlet, MA showed protonated molecule ions, [MA + H]+m/z 150. As analytes were introduced via inlet-1, the stability of the HCD background ion signal reduced, leading to a decrease in sensitivity. Unlike that in negative ion mode, introduction of ambient air in positive ion mode enhanced the sensitivity of the air HCD ion source through the formation of hydronium ions, which gave protonated molecule ions. Ionization mechanisms are also discussed. [ABSTRACT FROM AUTHOR]

2-Benzylbenzimidazoles, or "nitazenes", are a class of novel synthetic opioids (NSOs) that are increasingly being detected alongside fentanyl analogs and other opioids in drug overdose cases. Nitazenes can be 20× more potent than fentanyl but are not routinely tested for during postmortem or clinical toxicology drug screens; thus, their prevalence in drug overdose cases may be under-reported. Traditional analytical workflows utilizing liquid chromatography-tandem mass spectrometry (LC–MS/MS) often require additional confirmation with authentic reference standards to identify a novel nitazene. However, additional analytical measurements with ion mobility spectrometry (IMS) may provide a path toward reference-free identification, which would greatly accelerate NSO identification rates in toxicology laboratories. Presented here are the first IMS and collision cross section (CCS) measurements on a set of fourteen nitazene analogs using a structures for lossless ion manipulations (SLIM)-orbitrap MS. All nitazenes exhibited two high intensity baseline-separated IMS distributions, which fentanyls and other drug and druglike compounds also exhibit. Incorporating water into the electrospray ionization (ESI) solution caused the intensities of the higher mobility IMS distributions to increase and the intensities of the lower mobility IMS distributions to decrease. Nitazenes lacking a nitro group at the R1 position exhibited the greatest shifts in signal intensities due to water. Furthermore, IMS-MS/MS experiments showed that the higher mobility IMS distributions of all nitazenes possessing a triethylamine group produced fragment ions with m/z 72, 100, and other low intensity fragments while the lower mobility IMS distributions only produced fragment ions with m/z 72 and 100. The IMS, solvent, and fragmentation studies provide experimental evidence that nitazenes potentially exhibit three gas-phase protomers. The cyclic IMS capability of SLIM was also employed to partially resolve four sets of structurally similar nitazene isomers (e.g., protonitazene/isotonitazene, butonitazene/isobutonitazene/secbutonitazene), showcasing the potential of using high-resolution IMS separations in MS-based workflows for reference-free identification of emerging nitazenes and other NSOs. [ABSTRACT FROM AUTHOR]

Currently, glycopeptide quantitation is mainly based on relative quantitation due to absolute quantitation requiring isotope-labeled or standard glycopeptides which may not be commercially available or are very costly and time consuming to synthesize. To address this grand challenge, coulometric mass spectrometry (CMS), based on the combination of electrochemistry (EC) and mass spectrometry (MS), was utilized to quantify electrochemically active glycopeptides without the need of using standard materials. In this study, we studied tyrosine-containing glycopeptides, NYIVGQPSS-(β-GlcNAc)-TGNL-OH and NYSVPSS-(β-GlcNAc)-TGNL-OH, and successfully quantified them directly with CMS with a discrepancy of less than 5% between the CMS measured amount and the theoretical amount. Taking one step further, we applied this approach to quantify glycopeptides generated from the digestion of NIST mAb, a monoclonal antibody reference material. Through HILIC column separation, five N297 glycopeptides resulting from NIST mAb tryptic digestion were successfully separated and quantified by CMS for an absolute amount without the use of any standard materials. This study indicates the potential utility of CMS for quantitative proteomics research. [ABSTRACT FROM AUTHOR]

The identification and quantitation of plasmalogen glycerophospholipids is challenging due to their isobaric overlap with plasmanyl ether-linked glycerophospholipids, susceptibility to acid degradation, and their typically low abundance in biological samples. Trimethylation enhancement using diazomethane (TrEnDi) can be used to significantly enhance the signal of glycerophospholipids through the creation of quaternary ammonium groups producing fixed positive charges using 13C-diazomethane in complex lipid extracts. Although TrEnDi requires a strong acid for complete methylation, we report an optimized protocol using 10 mM HBF4 with the subsequent addition of a buffer solution that prevents acidic hydrolysis of plasmalogen species and enables the benefits of TrEnDi to be realized for this class of lipids. These optimized conditions were applied to aliquots of bovine liver extract (BLE) to achieve permethylation of plasmalogen lipids within a complex mixture. Treating aliquots of unmodified and TrEnDi-derivatized BLE samples with 80% formic acid and comparing their liquid chromatography mass spectrometry (LCMS) results to analogous samples not treated with formic acid, enabled the identification of 29 plasmalogen species. On average, methylated plasmalogen species from BLE demonstrated 2.81-fold and 28.1-fold sensitivity gains over unmodified counterparts for phosphatidylcholine and phosphatidylethanolamine plasmalogen species, respectively. Furthermore, the compatibility of employing 13C-TrEnDi and a previously reported iodoacetalization strategy was demonstrated to effectively identify plasmenyl-ether lipids in complex biological extracts at greater levels of sensitivity. Overall, we detail an optimized 13C-TrEnDi derivatization strategy that enables the analysis of plasmalogen glycerophospholipids with no undesired cleavage of radyl groups, boosting their sensitivity in LCMS and LCMS/MS analyses. [ABSTRACT FROM AUTHOR]

Traditionally, mass spectrometry (MS) output is the ion abundance plotted versus the ionic mass-to-charge ratio m/z. While employing only commercially available equipment, Charge Determination Analysis (CHARDA) adds a third dimension to MS, estimating for individual peaks their charge states z starting from z = 1 and color coding z in m/z spectra. CHARDA combines the analysis of ion signal decay rates in the time-domain data (transients) in Fourier transform (FT) MS with the interrogation of mass defects (fractional mass) of biopolymers. Being applied to individual isotopic peaks in a complex protein tandem (MS/MS) data set, CHARDA aids peptide mass spectra interpretation by facilitating charge-state deconvolution of large ionic species in crowded regions, estimating z even in the absence of an isotopic distribution (e.g., for monoisotopic mass spectra). CHARDA is fast, robust, and consistent with conventional FTMS and FTMS/MS data acquisition procedures. An effective charge-state resolution Rz ≥ 6 is obtained with the potential for further improvements. [ABSTRACT FROM AUTHOR]

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion–ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3′-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5′-GGGGGGGG-3′. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5′-AAAAAAAA-3′. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G. [ABSTRACT FROM AUTHOR]

The opioid crisis in the United States is being fueled by the rapid emergence of new fentanyl analogs and precursors that can elude traditional library-based screening methods, which require data from known reference compounds. Since reference compounds are unavailable for new fentanyl analogs, we examined if fentanyls (fentanyl + fentanyl analogs) could be identified in a reference-free manner using a combination of electrospray ionization (ESI), high-resolution ion mobility (IM) spectrometry, high-resolution mass spectrometry (MS), and higher-energy collision-induced dissociation (MS/MS). We analyzed a mixture containing nine fentanyls and W-15 (a structurally similar molecule) and found that the protonated forms of all fentanyls exhibited two baseline-separated IM distributions that produced different MS/MS patterns. Upon fragmentation, both IM distributions of all fentanyls produced two high intensity fragments, resulting from amine site cleavages. The higher mobility distributions of all fentanyls also produced several low intensity fragments, but surprisingly, these same fragments exhibited much greater intensities in the lower mobility distributions. This observation demonstrates that many fragments of fentanyls predominantly originate from one of two different gas-phase structures (suggestive of protomers). Furthermore, increasing the water concentration in the ESI solution increased the intensity of the lower mobility distribution relative to the higher mobility distribution, which further supports that fentanyls exist as two gas-phase protomers. Our observations on the IM and MS/MS properties of fentanyls can be exploited to positively differentiate fentanyls from other compounds without requiring reference libraries and will hopefully assist first responders and law enforcement in combating new and emerging fentanyls. [ABSTRACT FROM AUTHOR]

The microsynthesis of 32 dialkyl derivatives of ethylphosphonic acid and the same number of monoalkyl derivatives was carried out to perform comparative studies using gas chromatography combined with mass spectrometry in chemical ionization mode which is one of the analytical techniques recommended by the Organisation for the Prohibition of Chemical Weapons (OPCW). The huge number of possible representatives makes it difficult to have complete spectral libraries of all substances in this class. Therefore, we decided to synthesize and instrumentally analyze only representatives of the selected series of homologues in this work. The analysis of the obtained results allowed us to find the rules for predicting mass spectra and the factors determining the retention parameters. Symmetrical diesters and monoesters of ethylphosphonic acid were selected for this study. During the conducted experiments using chemical ionization with methane as the reaction gas, protonated analyte molecules with high relative intensities were obtained; in many cases, these are base peaks in the spectrum. The obtained results allow grouping of the synthesized compounds depending on the introduced alkyl substituent. Retention data of the tested analytes were collected during the research by using electron ionization. The retention parameters of the tested compounds from each homologous series were also summarized and compared. Chemical Warfare Agents (CWA) analysis continues to be an important issue, especially in the context of the regular Proficiency Tests organized by the OPCW for identifying chemical compounds that are of interest to the Chemical Weapons Convention. Five compounds were synthesized whose spectra were not available in EI mass spectral libraries, and their retention indices were unknown. The identification of these substances was supported by the CI mass spectra and retention data, using previously developed relationships. Therefore, it is reasonable to conclude that the research method used is useful and effective. [ABSTRACT FROM AUTHOR]

Over the past century, agriculture practices have transitioned from manual cultivation to the use of an array of chemical herbicides for weed control including phosphinothricin, or glufosinate (GLUF). Consequently, the potential for long-term residual GLUF exposure in the food chain has increased, highlighting the need for improved analytical strategies for its detection, as well as the detection of its main breakdown product 3-(methylphosphinico)-propionic acid (MPPA). Chemical derivatization strategies have been developed to improve the detection of GLUF and MPPA via liquid chromatography tandem mass spectrometry analyses. Herein, we employ trimethylation enhancement using diazomethane (TrEnDi) for the first time as a means to confer analytical advantages via quantitatively derivatizing these analytes into permethylated GLUF ([GLUFTr]+) and MPPA ([MPPATr+H]+). Comparing [GLUFTr]+ and [MPPATr+H]+ to underivatized counterparts, TrEnDi yields 2.8-fold and 1.7-fold improvements in reversed-phase chromatographic retention, respectively, while MS-based sensitivity is enhanced 4.1-fold and 11.0-fold, respectively. Successful analyte derivatization (with >99% yields) was further demonstrated on a commercial herbicide solution imparting consistent analytical enhancements. To investigate the benefits of TrEnDi in a bona fide agricultural scenario, simple aqueous extractions from distinct parts of field-grown canola plants were performed to quantify GLUF and MPPA before and after TrEnDi derivatization. In their underivatized forms, GLUF and MPPA were undetectable in all field samples, whereas [GLUFTr]+ and [MPPATr+H]+ were readily quantifiable using the same analysis conditions. Our results demonstrate that TrEnDi continues to be a useful tool to enhance the analytical characteristics of organic molecules that are traditionally difficult to detect. [ABSTRACT FROM AUTHOR]

13C-Trimethylation enhancement using diazomethane (13C-TrEnDi) is a chemical derivatization technique that uses 13C-labeled diazomethane to increase mass spectrometry (MS) signal intensities for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid classes, both of which are of major interest in biochemistry. In silico mass spectrometry databases have become mainstays in lipidomics experiments; however, 13C-TrEnDi-modified PC and PE species have altered m/z and fragmentation patterns from their native counterparts. To build a database of 13C-TrEnDi-modified PC and PE species, a lipid extract from nutritional yeast was derivatized and fragmentation spectra of modified PC and PE species were mined using diagnostic fragmentation filtering by searching 13C-TrEnDi-modified headgroups with m/z 199 (PC) and 202 (PE). Identities of 25 PC and 10 PE species were assigned after comparing to predicted masses from the Lipid Maps Structure Database with no false positive identifications observed; neutral lipids could still be annotated after derivatization. Collision energies from 16 to 52 eV were examined, resulting in three additional class-specific fragment ions emerging, as well as a combined sn-1/sn-2 fragment ion, allowing sum-composition level annotations to be assigned. Using the Lipid Blast templates, a NIST-compatible 13C-TrEnDi database was produced based on fragmentation spectra observed at 36 eV and tested on HEK 293T cell lipid extracts, identifying 47 PC and 24 PE species, representing a 1.8-fold and 2.2-fold increase in annotations, respectively. The 13C-TrEnDi database is freely available, MS vendor-independent, and widely compatible with MS data processing pipelines, increasing the throughput and accessibility of TrEnDi for lipidomics applications. [ABSTRACT FROM AUTHOR]

Although molecular analysis and imaging by mass spectrometry are emerging as tools to identify metabolites and determine their distribution in cells and tissues, it is difficult to directly analyze the labile molecules at the single-cell level. Glucosinolate (GL) is a plant-active substance with much attention as a chemical defense mechanism known as a "mustard oil bomb" in broccoli. When tissue is damaged, these substances undergo rapid degradation, making them unsuitable for conventional mass spectrometry (MS), particularly for surface MS imaging analysis methods that necessitate intricate preprocessing. Herein, a strategy combining cryogenic laser ablation inductively coupled mass spectrometry (CLA-ICP-MS) and capillary microsampling nanospray high-resolution mass spectrometry (HRMS) was developed. The sulfur-rich microzone in tissue which was thought as a suspect GL-rich cell population was located via CLA-ICP-MS. Three GLs in single cells were accurately identified by nanospray HRMS with a hydrogen/deuterium exchange reaction. Subsequently, cell-by-cell imaging by nanospray HRMS showed that the GL-rich cells were below the stalk surface by approximately 30 μm. This proposed strategy can also be applied to rapidly identify labile compounds and localize molecule-rich cells in tissues. [ABSTRACT FROM AUTHOR]

Meningiomas are among the most common brain tumors that arise from the leptomeningeal cover of the brain and spinal cord and account for around 37% of all central nervous system tumors. According to the World Health Organization, meningiomas are classified into three histological subtypes: benign, atypical, and anaplastic. Sometimes, meningiomas with a histological diagnosis of benign tumors show clinical characteristics and behavior of aggressive tumors. In this study, we examined the metabolomic and lipidomic profiles of meningioma tumors, focusing on comparing low-grade and high-grade tumors and identifying potential markers that can discriminate between benign and malignant tumors. High-resolution mass spectrometry coupled to liquid chromatography was used for untargeted metabolomics and lipidomics analyses of 85 tumor biopsy samples with different meningioma grades. We then applied feature selection and machine learning techniques to find the features with the highest information to aid in the diagnosis of meningioma grades. Three biomarkers were identified to differentiate low- and high-grade meningioma brain tumors. The use of mass-spectrometry-based metabolomics and lipidomics combined with machine learning analyses to prospect and characterize biomarkers associated with meningioma grades may pave the way for elucidating potential therapeutic and prognostic targets. [ABSTRACT FROM AUTHOR]

Glycosidic linkages in oligosaccharides play essential roles in determining their chemical properties and biological activities. MSn has been widely used to infer glycosidic linkages but requires a substantial amount of starting material, which limits its application. In addition, there is a lack of rigorous research on what MSn protocols are proper for characterizing glycosidic linkages. In this work, to deliver high-quality experimental data and analysis results, we propose a machine learning-based framework to establish appropriate MSn protocols and build effective data analysis methods. We demonstrate the proof-of-principle by applying our approach to elucidate sialic acid linkages (α2′–3′ and α2′–6′) in a set of sialyllactose standards and NIST sialic acid-containing N-glycans as well as identify several protocol configurations for producing high-quality experimental data. Our companion data analysis method achieves nearly 100% accuracy in classifying α2′–3′ vs α2′–6′ using MS5, MS4, MS3, or even MS2 spectra alone. The ability to determine glycosidic linkages using MS2 or MS3 is significant as it requires substantially less sample, enabling linkage analysis for quantity-limited natural glycans and synthesized materials, as well as shortens the overall experimental time. MS2 is also more amenable than MS3/4/5 to automation when coupled to direct infusion or LC-MS. Additionally, our method can predict the ratio of α2′–3′ and α2′–6′ in a mixture with 8.6% RMSE (root-mean-square error) across data sets using MS5 spectra. We anticipate that our framework will be generally applicable to analysis of other glycosidic linkages. [ABSTRACT FROM AUTHOR]

Highly selective enrichment of low abundance N-glyco- and phosphopeptides from intricate biological samples before mass spectrometry detection is a vital prerequisite for in-depth proteomics. A hydrophilic porous magnetic graphene oxide/chitosan composite (mGO/CS beads) was developed and applied to selectively enrich N-glyco- and phosphopeptides in biological samples. The obtained mGO/CS beads showed strong hydrophilicity, large pore structure, and magnetic property. Based on the multivalent hydrophilic interactions between glycan group on N-glycopeptides, phosphate on phosphopeptides, and amino and hydroxyl groups on chitosan, N-glyco- and phosphopeptides could be simultaneously captured by mGO/CS beads and sequentially released by different eluents. The mGO/CS beads exhibited high sensitivity (limit of detection: 1.0 fmol μL–1 for N-glycopeptides and 0.5 fmol μL–1 for phosphopeptides) and high selectivity (IgG/BSA and β-casein/BSA tryptic digests with a mass ratio of 1:100 and 1:200). After using mGO/CS beads treatment, a total of 83 N-glycopeptides assigned to 80 N-glycoproteins and 10 phosphopeptides were identified by mass spectrometry from human serum and nonfat milk, respectively. These experimental results indicated that mGO/CS beads have great potential for highly efficient enrichment of low-abundance N-glyco- and phosphopeptides in complex biological samples. [ABSTRACT FROM AUTHOR]

Due to its speed, accuracy, and adaptability to various sample types, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a popular method to identify molecular isotope profiles from biological samples. Often MALDI-MS data do not include tandem MS fragmentation data, and thus the identification of compounds in samples requires external databases so that the accurate mass of detected signals can be matched to known molecular compounds. Most relevant MALDI-MS software tools developed to confirm compound identifications are focused on small molecules (e.g., metabolites, lipids) and cannot be easily adapted to protein data due to their more complex isotopic distributions. Here, we present an R package called IsoMatchMS for the automated annotation of MALDI-MS data for multiple datatypes: intact proteins, peptides, and glycans. This tool accepts already derived molecular formulas or, for proteomics applications, can derive molecular formulas from a list of input peptides or proteins including proteins with post-translational modifications. Visualization of all matched isotopic profiles is provided in a highly accessible HTML format called a trelliscope display, which allows users to filter and sort by several parameters such as match scores and the number of peaks matched. IsoMatchMS simplifies the annotation and visualization of MALDI-MS data for downstream analyses. [ABSTRACT FROM AUTHOR]

Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization. [ABSTRACT FROM AUTHOR]

Photoionization schemes for mass spectrometry, either by laser or discharge lamps, have been widely examined and deployed. In this work, the ionization characteristics of a xenon discharge lamp (Xe-APPI, 9.6/8.4 eV) have been studied and compared to established ionization schemes, such as atmospheric pressure chemical ionization, atmospheric pressure photoionization with a krypton discharge lamp (Kr-APPI, 10.6/10 eV) and atmospheric pressure laser ionization (266 nm). Addressing the gas-phase ionization behavior has been realized by gas chromatography coupled to high-resolution mass spectrometry without the usage of a dopant. For standard substances, it has been found that Xe-APPI is able to ionize a broad range of polycyclic aromatic hydrocarbons as well as their heteroatom-containing and alkylated derivatives. However, thiol and ester compounds could not be detected. Moreover, Xe-APPI revealed a high tendency to generate oxygenated artifacts, most likely due to a VUV absorption band of oxygen at 148 nm. Beneficially, almost no chemical background, commonly caused by APCI or Kr-APPI due to column blood, plasticizers or impurities, is observed. This advantage is noteworthy for evolved gas analysis without preseparation or for chromatographic coelution. For the complex mixtures, Xe-APPI revealed the predominant generation of radical cations via direct photoionization with a high selectivity toward aromatic core structures with low alkylation. Interestingly, both Xe-APPI and Kr-APPI could sensitively detect sterane cycloalkanes, validated by gas chromatographic retention. The narrowly ionized chemical space could let Xe-APPI find niche applications, e.g., for strongly contaminated samples to reduce the background. [ABSTRACT FROM AUTHOR]

Lipid peroxidation is a key component in the pathogenesis of numerous disease states, where the oxidative damage of lipids frequently leads to membrane dysfunction and subsequent cellular death. Glycerophospho-ethanolamine (PE) is the second most abundant phospholipid found in cellular membranes and, when oxidized, has been identified as an executor of ferroptotic cell death. PE commonly exists in the plasmalogen form, where the presence of the vinyl ether bond and its enrichment in polyunsaturated fatty acids make it especially susceptible to oxidative degradation. This results in a multitude of oxidized products complicating identification and often requiring several analytical techniques for interpretation. In the present study, we outline an analytical approach for the structural characterization of intact oxidized products of arachidonate-containing diacyl and plasmalogen PE. Intact oxidized PE structures, including structural and positional isomers, were identified using complementary liquid chromatography techniques, drift tube ion mobility, and high-resolution tandem mass spectrometry. This work establishes a comprehensive method for the analysis of intact lipid peroxidation products and provides an important pathway to investigate how lipid peroxidation initially impacts glycerophospholipids and their role in redox biology. [ABSTRACT FROM AUTHOR]

Combinatorial post-translational modifications (PTMs), such as those forming the so-called "histone code", have been linked to cell differentiation, embryonic development, cellular reprogramming, aging, cancers, neurodegenerative disorders, etc. Nevertheless, a reliable mass spectral analysis of the combinatorial isomers represents a considerable challenge. The difficulty stems from the incompleteness of information that could be generated by the standard MS to differentiate cofragmented isomeric sequences in their naturally occurring mixtures based on the fragment mass-to-charge ratio and relative abundance information only. Here we show that fragment–fragment correlations revealed by two-dimensional partial covariance mass spectrometry (2D-PC-MS) allow one to solve the combinatorial PTM puzzles that cannot be tackled by the standard MS as a matter of principle. We introduce 2D-PC-MS marker ion correlation approach and demonstrate experimentally that it can provide the missing information enabling identification of cofragmentated combinatorially modified isomers. Our in silico study shows that the marker ion correlations can be used to unambiguously identify 5 times more cofragmented combinatorially acetylated tryptic peptides and 3 times more combinatorially modified Glu-C peptides of human histones than is possible using standard MS methods. [ABSTRACT FROM AUTHOR]

Proteomics research has been transformed due to high-throughput liquid chromatography (LC-MS/MS) tandem mass spectrometry instruments combined with highly sophisticated automated sample preparation and multiplexing workflows. However, scaling proteomics experiments to large sample cohorts (hundreds to thousands) requires thoughtful quality control (QC) protocols. Robust QC protocols can help with reproducibility, quantitative accuracy, and provide opportunities for more decisive troubleshooting. Our laboratory conducted a plasma proteomics study of a cohort of N = 335 patient samples using tandem mass tag (TMTpro) 16-plex batches. Over the course of a 10-month data acquisition period for this cohort we collected 271 pooled QC LC-MS/MS result files obtained from MS/MS analysis of a patient-derived pooled plasma sample, representative of the entire cohort population. This sample was tagged with TMTzero or TMTpro reagents and used to inform the daily performance of the LC-MS/MS instruments and to allow within and across sample batch normalization. Analytical variability of a number of instrumental and data analysis metrics including protein and peptide identifications, peptide spectral matches (PSMs), number of obtained MS/MS spectra, average peptide abundance, percent of peptides with a Δ m/z between ±0.003 Da, percent of MS/MS spectra obtained at the maximum injection time, and the retention time of selected tracking peptides were evaluated to help inform the design of a robust LC-MS/MS QC workflow for use in future cohort studies. This study also led to general tips for using selected metrics to inform real-time troubleshooting of LC-MS/MS performance issues with daily QC checks. [ABSTRACT FROM AUTHOR]

Glyphosate (GLY), a synthetic, nonselective systemic herbicide that is particularly effective against perennial weeds, is the most used weedkiller in the world. There are growing concerns over GLY accumulation in the environment and the attendant human health-associated risks, and despite increased attention in the media, GLY and its breakdown product aminomethylphosphonic acid (AMPA) remain elusive to many analytical strategies. Chemical derivatization coupled with high-performance liquid chromatography-mass spectrometry (HPLC-MS) addresses the challenge of quantifying low levels of GLY and AMPA in complex samples. Here we demonstrate the use of in situ trimethylation enhancement using diazomethane (iTrEnDi) to derivatize GLY and AMPA into permethylated products ([GLYTr]+ and [AMPATr]+, respectively) prior to analysis via HPLC-MS. iTrEnDi produced quantitative yields and resulted in a 12–340-fold increases in HPLC-MS-based sensitivity for [GLYTr]+ and [AMPATr]+, respectively, compared with underivatized counterparts. The limits of detection of derivatized compounds were found to be 0.99 ng/L for [GLYTr]+ and 1.30 ng/L for [AMPATr]+, demonstrating significant sensitivity improvements compared to previously established derivatization techniques. iTrEnDi is compatible with the direct derivatization of Roundup formulations. Finally, as proof of principle, a simple aqueous extraction followed by iTrEnDi enabled the detection of [GLYTr]+ and [AMPATr]+ on the exterior of field-grown soybeans that were sprayed with Roundup. Overall, iTrEnDi ameliorates issues relating to low proton affinity and chromatographic retention, boosting HPLC-MS-based sensitivity and enabling the elucidation of elusive analytes such as GLY and AMPA within agricultural systems. [ABSTRACT FROM AUTHOR]

To capture the structure of assembled hemagglutinin (HA) nanoparticles at single-particle resolution, HA-specific antigen binding fragments (Fabs) were labeled by fluorescent (FLR) dyes as probes to highlight the HA trimers displayed on the assembled tetravalent HA nanoparticles for a qualitative localization microscopic study. The FLR dyes were conjugated to the Fabs through N-hydroxysuccinimide (NHS) ester mediated amine coupling chemistry. The labeling profile, including labeling ratio, distribution, and site-specific labeling occupancy, can affect the imaging results and introduce inconsistency. To evaluate the labeling profile so as to evaluate the labeling efficiency, a combination of intact mass measurement by MALDI-MS and peptide mapping through LC-MS/MS was implemented. At the intact molecular level, the labeling ratio and distribution were determined. Through peptide mapping, the labeled residues were identified and the corresponding site-specific labeling occupancy was measured. A systematic comparative investigation of four different FLR-labeled 1H01-Fabs (generated from H1 strain HA specific mAb 1H01) allowed accurate profiling of the labeling pattern. The data indicate that the labeling was site-specific and semiquantitative. This warrants the consistency of single-particle fluorescent imaging experiments and allows a further imaging characterization of the single nanoparticles. [ABSTRACT FROM AUTHOR]

As part of a larger effort to aid in seamless integration of Fourier-based multiplexed ion mobility with a range mass analyzers, we have developed an all-in-one graphical user interface tool for FT-IM-MS data analysis that runs directly within a web browser. This tool, FTflow, accepts mzML files and displays necessary information such as mass spectra and extracted ion chromatograms in order to reconstruct arrival time distributions. It also extracts the corresponding mobility-related information (e.g., Ko and CCS) for each of the target ion populations. Furthermore, input fields for experimental conditions are clearly laid out for users and ease-of-use. With flexibility in mind, the processing scripts and GUI interface are written entirely in Python and allows users the option to modify source code to fit their specific needs. While the intention for this tool is to be a starting point for exploratory analysis of FT-IM-MS data, it has the capability to be adapted for use in more automated data processing pipelines through direct access of core processing routines. [ABSTRACT FROM AUTHOR]

Over 2.5 million neonatal dried blood spots (DBS) are stored at the Danish National Biobank. These samples offer extraordinary possibilities for metabolomics research, including prediction of disease and understanding of underlying molecular mechanisms of disease development. Nevertheless, Danish neonatal DBS have been little explored in metabolomics studies. One question that remains underinvestigated is the long-term stability of the large number of metabolites typically assessed in untargeted metabolomics over long time periods of storage. Here, we investigate temporal trends of metabolites measured in 200 neonatal DBS collected over a time course of 10 years, using an untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) based metabolomics protocol. We found that a majority (71%) of the metabolome was stable during 10 years of storage at −20 °C. However, we found decreasing trends for lipid-related metabolites, such as glycerophosphocholines and acylcarnitines. A few metabolites, including glutathione and methionine, may be strongly influenced by storage, with changes in metabolite levels up to 0.1–0.2 standard deviation units per year. Our findings indicate that untargeted metabolomics of DBS samples, with long-term storage in biobanks, is suitable for retrospective epidemiological studies. We identify metabolites whose stability in DBS should be closely monitored in future studies of DBS samples with long-term storage. [ABSTRACT FROM AUTHOR]

While soluble forms of amyloid-β (Aβ) and Tau work together to drive healthy neurons into a disease state, how their interaction may control the prion-like propagation and neurotoxicity of Tau is not fully understood. The cross-linking via disulfide bond formation is crucial for Tau oligomers to obtain stable conformers and spread between cells. This work thus focuses on how Aβ42 regulates this critical process. By studying the interactions between Aβ42 and TauPHF43, a construct that mimics the Tau R3 isoform, has a similar length to Aβ42, and contains one cysteine (Cys-322), we discovered that fresh Aβ42 could protect Tau against the formation of disulfide cross-linked dimers. We showed that the monomeric and small Aβ oligomers (the "nonamyloidogenic Aβ") efficiently disassembled tau dimers and heparin-induced Tau oligomers to recover Tau monomers. Interestingly, Aβ serves the role of an antioxidant to prevent disulfide bond formation, as supported by the experiments of Aβ with cystine. Furthermore, using cyclosporine A (CycA), a macrocyclic β-sheet disruptor, we demonstrated that targeting amyloidogenic Aβ with CycA does not affect the TauPHF43 disassembly driven by Aβ42. Separately, we assessed the initial toxicity of Aβ42 and TauPHF43 in acute brain slices and found that Aβ42 is more toxic than TauPHF43 or the two peptides combined. Our work highlights a potential protective role of Aβ42 monomers in AD that was previously overlooked while focusing on the mechanism behind Aβ42 aggregation leading to tau dysfunction. [ABSTRACT FROM AUTHOR]

In sheep, the transmissibility and progression of scrapie, a sheep prion (PrPSc) disease, is strongly dependent upon specific amino acid polymorphisms in the natively expressed prion protein (PrPC). Sheep expressing PrPC with lysine (K) polymorphism at position 171 (K171) are partially resistant to oronasal dosing of classical sheep scrapie. In addition, scrapie infected sheep expressing the K171 polymorphism show a longer incubation period compared to sheep homozygous (glutamine (Q)) at position 171. Quantitating the amount of the K171 polymorphism in a sheep scrapie sample can provide important information on the composition of PrPSc. A tryptic peptide, 159R.YPNQVYYRPVDK.Y172, derived from the digestion of 171K recombinant PrP, was identified as an analyte peptide suitable for a multiple reaction monitoring-based analysis. This method, using 15N-labeled analogs and another internal peptide from the proteinase K-resistant core, permits the simultaneous quantitation of the total amount of PrP and the proportion of K171 polymorphism in the sample. Background molecules with similar retention times and transitions were present in samples from scrapie-infected sheep. Proteinase K digestion followed by ultracentrifugation-based isolation or phosphotungstic acid-based isolation were employed to minimize the contribution of those background molecules, making this approach suitable for quantitating the amount of the K171 polymorphism in heterozygous scrapie infected sheep. [ABSTRACT FROM AUTHOR]

The normal cellular prion protein (PrPC) and its infectious conformer, PrPSc, possess a disproportionately greater amount of methionines than would be expected for a typical mammalian protein. The thioether of methionine can be readily oxidized to the corresponding sulfoxide, which means that oxidation of methionine can be used to map the surface of the conformation of PrPC or PrPSc, as covalent changes are retained after denaturation. We identified a set of peptides (TNMK, MLGSAMSR, LLGSAMSR, PMIHFGN-DWEDR, ENMNR, ENMYR, IMER, MMER, MIER, VVEQMC-VTQYQK, and VVEQMCI-TQYQR) that contains every methionine in sheep, cervid, mouse, and bank vole PrP. Each is the product of a tryptic digestion and is suitable for a multiple reaction monitoring (MRM) based analysis. The peptides chromatograph well. The oxidized and unoxidized peptides containing one methionine readily separate. The unoxidized, two singly oxidized, and doubly oxidized forms of the MLGSAMSR and MMER peptides are also readily distinguishable. This approach can be used to determine the surface exposure of each methionine by measuring its oxidation after reaction with added hydrogen peroxide. [ABSTRACT FROM AUTHOR]

With Intact Transition Epitope Mapping-Thermodynamic Weak-force Order (ITEM-TWO) analysis in combination with molecular modeling, the phosphorylation-dependent molecular recognition motif of the anti-HpTGEKP antibody has been investigated with binary and ternary component mixtures consisting of antibody and (phospho-) peptides. Amino acid sequences have been selected to match either the antibody's recognition motif or the cancer-related zinc finger protein mutations and phosphorylations of the respective amino acid residues. Upon electrospraying of all the components of the mixtures, that is, hexapeptides, antibody, and intact immune complexes, the produced ions were subjected to mass spectrometric mass filtering. The antibody ions as well as the immune complex ions traversed into the mass spectrometer's collision chamber, whereas paths of unbound peptide ions were blocked prior to entering the collision cell. After dissociation of the multiply charged immune complexes in the gas phase, the complex-released peptide ions were recorded after having traversed the second mass filter. Complex-released peptides were unambiguously identified by their masses using mass analysis with isotope resolution. From the results of our studies with seven (phospho-) peptides with distinct amino acid sequences, which resembled either the antibody's binding motif or mutations, we conclude the following: (i) A negatively charged phospho group, located near the peptide's N-terminus is mandatory for antibody binding when placed on the peptide surface at a precise distance to the C-terminally located positively charged ε-amino group of a lysinyl residue. (ii) A bulky amino acid residue, such as the tyrosinyl residue at the N-terminal position of the (phospho-) threoninyl residue, abolishes antibody binding. (iii) Two closely spaced phospho groups negatively interfere with the surface polarity pattern and abolish antibody binding as well. (iv) Non-phosphorylated peptides are not binding partners of the anti-HpTGEKP antibody. [ABSTRACT FROM AUTHOR]

Mass spectrometry-based discovery glycoproteomics is highly dependent on the use of chromatography paradigms amenable to analyte retention and separation. When compared against established stationary phases such as reversed-phase and hydrophilic interaction liquid chromatography, reports utilizing porous graphitic carbon have detailed its numerous advantages. Recent efforts have highlighted the utility in porous graphitic carbon in high-throughput glycoproteomics, principally through enhanced profiling depth and liquid-phase resolution at higher column temperatures. However, increasing column temperature has been shown to impart disparaging effects in glycopeptide identification. Herein we further elucidate this trend, describing qualitative and semiquantitative effects of increased column temperature on glycopeptide identification rates, signal intensity, resolution, and spectral count linear response. Through analysis of enriched bovine and human glycopeptides, species with high mannose and sialylated glycans were shown to most significantly benefit and suffer from high column temperatures, respectively. These results provide insight as to how porous graphitic carbon separations may be appropriately leveraged for glycopeptide identification while raising concerns over quantitative and semiquantitative label-free comparisons as the temperature changes. RAW MS glycoproteomic data are available via ProteomeXchange with identifier PXD034354. [ABSTRACT FROM AUTHOR]

Design-of-experiment (DOE) approaches, originally conceived by Fischer, are widely applied in industry, particularly in the context of production for which they have been greatly expended. In a research and development context, DOE can be of great use for method development. Specifically, DOE can greatly speed up instrument parameter optimization by first identifying parameters that are critical to a given outcome, showing parameter interdependency where it occurs and accelerating optimization of said parameters using matrices of experimental conditions. While DOE approaches have been applied in mass spectrometry experiments, they have so far failed to gain widespread adoption. This could be attributed to the fact that DOE can get quite complex and daunting to the everyday user. Here we make the case that a subset of DOE tools, hereafter called SimpleDOE (sDOE), can make DOE accessible and useful to the Mass Spectrometry community at large. We illustrate the progressive gains from a purely manual approach to sDOE through a stepwise optimization of parameters affecting the efficiency of top-down ETD fragmentation of proteins on a high-resolution Q-TOF mass spectrometer, where the aim is to maximize sequence coverage of fragmentation events. [ABSTRACT FROM AUTHOR]

Identification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel antibody formats. As a standard analytical approach, manual offline fractionation of charge variants by cation-exchange chromatography followed by comprehensive analytical testing is applied. These conventional workflows are time-consuming and labor-intensive and overall reach their limits in terms of chromatographic separation of enhanced structural heterogeneities raised from new antibody formats. For these reasons, we aimed to develop an alternative online characterization strategy for charge variant characterization of a therapeutic bispecific antibody by online mD-LC-MS at middle-up (2D-LC-MS) and bottom-up (4D-LC-MS) level. Using the implemented online mD-LC-MS approach, all medium- and even low-abundant product variants previously identified by offline fraction experiments and liquid chromatography mass spectrometry could be monitored. The herein reported automated online mD-LC-MS methodology therefore represents a complementary and in part alternative approach for analytical method validation including multiattribute monitoring (MAM) strategies by mass spectrometry, offering various benefits including increased throughput and reduced sample handling and combined protein information at intact protein and peptide level. [ABSTRACT FROM AUTHOR]

Sub-nanometer gold clusters are promising size- and composition-tunable materials that may be used for advanced technological applications such as catalysis, energy generation, and microelectronics. Synthesis and characterization of phosphine ligated gold clusters containing different ligands provide insight into how steric and electronic effects resulting from changes in chemical functionality influence cluster size, stability, and formation in solution. Herein, we demonstrate that synthesizing gold clusters using two different diphosphines in solution at the same time results in a broad distribution of novel mixed-ligand clusters. In comparison, adding a second diphosphine to a solution of gold clusters presynthesized with another diphosphine does not result in extensive formation of mixed-ligand species. Utilizing high-mass resolution electrospray ionization mass spectrometry, we determined novel cluster compositions and observed size-dependent trends in gold clusters that undergo ligand exchange forming mixed diphosphine species. Adjacent peaks in the mass spectra, separated by characteristic mass-to-charge ratios, provide evidence for multiple 1,3-bis-(diphenylphosphino)-propane (L3) and 1,5-bis-(diphenylphosphino)-pentane (L5) ligands on cationic clusters containing 8, 10, 11, and 22 gold atoms. Energy-resolved collision-induced dissociation experiments provide qualitative insight into how different diphosphine ligands affect the relative stability of specific size gold clusters. Our results indicate that mixed-ligand clusters containing both L3 and L5 are generally more stable than their single ligand counterparts containing either L3 or L5. These molecular-level insights will facilitate the rational and scalable synthesis of gold clusters for targeted applications. [ABSTRACT FROM AUTHOR]

Protein glycosylation, covalent attachment of carbohydrates to polypeptide chains, is a highly important post-translational modification involved in many essential physiological processes. Comprehensive site-specific and quantitative analysis is crucial for revealing the diverse functions and dynamics of glycosylation. To characterize intact glycopeptides, mass spectrometry (MS)-based glycoproteomics employs versatile fragmentation methods, among which electron-transfer/higher-energy collision dissociation (EThcD) has gained great popularity. However, the inherent limitation of EThcD in fragmenting low-charge ions has prevented its widespread applications. Furthermore, there is a need to develop a high-throughput strategy for comparative glycoproteomics with a large cohort of samples. Herein, we developed isobaric N,N-dimethyl leucine-derivatized ethylenediamine (DiLeuEN) tags to increase the charge states of glycopeptides, thereby improving the fragmentation efficiency and allowing for in-depth intact glycopeptide analysis, especially for sialoglycopeptides. Moreover, the unique reporter ions of DiLeuEN-labeled glycopeptides generated in tandem MS spectra enable relative quantification of up to four samples in a single analysis, which represents a new high-throughput method for quantitative glycoproteomics. [ABSTRACT FROM AUTHOR]

By casting the information regarding an ion population's mobility in the frequency domain, the coupling of time-dispersive ion mobility techniques is now imminently compatible with slower mass analyzers such as ion traps. Recent reports have detailed the continued progress toward maximizing the efficiency of the Fourier transform ion mobility-mass spectrometry (FT-IM-MS) experiments, but few reports have outlined the intersection between the practical considerations of implementation against the theoretical limits imposed by traditional signal processing techniques. One of the important concerns for Fourier-based multiplexing experiments is avoiding signal aliasing as a product of undersampled signals that may occur during data acquisition. In addition to traditional considerations such as detector sampling frequency, the limitations (i.e., maximum measurable drift time) imposed by experimental mass scan duration and the frequency sweep used for ion gate modulation must also be assessed. This work aims to connect the fundamental underpinnings of FT-IM-MS experiments and the associated experimental parameters that are encountered when coupling the comparatively fast separations in the mobility domain with the slower m/z scanning common for ion-trap mass analyzers. In addition to stating the relevant theory that applies to the FT-IM-MS experiment, this report highlights how aliased signals will manifest post Fourier transform in reconstructed arrival time distributions and calculated mobilities. [ABSTRACT FROM AUTHOR]

Chromatographic-less mass spectrometry techniques like direct analysis in real-time mass spectrometry (DART-MS) are steadily being employed as seized drug screening tools. However, these newer analytical platforms require new computational methods to best make use of the collected data. The inverted library search algorithm (ILSA) is a recently developed method designed specifically for working with mass spectra of mixtures collected with DART-MS and has been implemented as a function in the NIST/NIJ DART-MS data interpretation tool (DIT). This paper demonstrates how DART-MS and the ILSA/DIT can be used to analyze seized drug evidence, while discussing insights gathered during the evaluation of 92 adjudicated case samples. The evaluation verified that the combination of DART-MS and the ILSA/DIT can be used as an informative tool to help analysts screen seized drug evidence but also revealed several factorssuch as the influence of incorporating multiple in-source fragmentation spectra and the effect of scoring thresholdsan analyst must consider while employing these methods. Use cases demonstrating the benefit of the nonscoring metrics provided by the ILSA/DIT and demonstrating how the ILSA/DIT can be used to identify novel substances are also presented. A summary of considerations for using the ILSA/DIT for drug screening concludes this paper. [ABSTRACT FROM AUTHOR]

Spectrum alignment of tandem mass spectrometry (MS/MS) data using the modified cosine similarity and subsequent visualization as molecular networks have been demonstrated to be a useful strategy to discover analogs of molecules from untargeted MS/MS-based metabolomics experiments. Recently, a neutral loss matching approach has been introduced as an alternative to MS/MS-based molecular networking with an implied performance advantage in finding analogs that cannot be discovered using existing MS/MS spectrum alignment strategies. To comprehensively evaluate the scoring properties of neutral loss matching, the cosine similarity, and the modified cosine similarity, similarity measures of 955 228 peptide MS/MS spectrum pairs and 10 million small molecule MS/MS spectrum pairs were compared. This comparative analysis revealed that the modified cosine similarity outperformed neutral loss matching and the cosine similarity in all cases. The data further indicated that the performance of MS/MS spectrum alignment depends on the location and type of the modification, as well as the chemical compound class of fragmented molecules. [ABSTRACT FROM AUTHOR]

Recent improvements in proteomics technologies have fundamentally altered our capacities to characterize human biology. There is an ever-growing interest in using these novel methods for studying the circulating proteome, as blood offers an accessible window into human health. However, every methodological innovation and analytical progress calls for reassessing our existing approaches and routines to ensure that the new data will add value to the greater biomedical research community and avoid previous errors. As representatives of HUPO's Human Plasma Proteome Project (HPPP), we present our 2024 survey of the current progress in our community, including the latest build of the Human Plasma Proteome PeptideAtlas that now comprises 4608 proteins detected in 113 data sets. We then discuss the updates of established proteomics methods, emerging technologies, and investigations of proteoforms, protein networks, extracellualr vesicles, circulating antibodies and microsamples. Finally, we provide a prospective view of using the current and emerging proteomics tools in studies of circulating proteins.

Owing to the role of the 20S proteasome in a wide spectrum of pathologies, including neurodegenerative disorders, proteasome-associated autoinflammatory syndromes (PRAAS), and cardiovascular diseases, understanding how its structure and composition contribute to dysfunction is crucial. As a 735 kDa protein assembly, the 20S proteasome facilitates normal cellular proteostasis by degrading oxidized and misfolded proteins. Declined proteasomal activity, which can be attributed to perturbations in the structural integrity of the 20S proteasome, is considered one of the main contributors to multiple proteasome-related diseases. Devising methods to characterize the structures of 20S proteasomes provides necessary insight for the development of drugs and inhibitors that restore proper proteasomal function. Here, native mass spectrometry was combined with multiple dissociation techniques, including ultraviolet photodissociation (UVPD), to identify the protein subunits comprising the 20S proteasome. UVPD, demonstrating an ability to uncover structural features of large (>300 kDa) macromolecular complexes, provided complementary information to conventional collision-based methods. Additionally, variable-temperature electrospray ionization was combined with UV photoactivation to study the influence of solution temperature on the stability of the 20S proteasome.

In modern biomedical research, cultivable cell lines are an indispensable tool, and the selection of cell lines that exhibit specific functional profiles is often critical to success. Cellular functional pathways have evolved through the selection of protein intra- and intermolecular interactions collectively referred to as the interactome. In the present work, quantitative in vivo protein cross-linking and mass spectrometry were used to probe large-scale protein interactome differences among three commonly employed human cell lines, namely, HEK293, MCF-7, and HeLa cells. These data illustrated highly reproducible quantitative interactome levels with R 2 values larger than 0.8 for all biological replicates. Proteome abundance levels were also measured using data-independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteome information allowed the visualization of cell type-specific interactome changes mediated by proteome level adaptations and independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the largest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study system-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight into cellular functional landscapes.

Covalent protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods identifying and quantifying covalent adducts in complex matrices. This study presents a workflow that combines data-dependent and data-independent acquisition (DDA and DIA) based liquid chromatography with tandem mass spectrometry (LC-MS/MS) to detect very low abundance adducts resulting from CYP mediated drug metabolism in human liver microsomes (HLMs). HLMs were incubated with raloxifene as a model compound and adducts were detected in 78 proteins, including CYP3A and CYP2C family enzymes. Experiments with recombinant CYP3A and CYP2C enzymes confirmed adduct formation in all CYPs tested, including CYPs not subject to time-dependent inhibition by raloxifene. These data suggest adducts can be benign. DIA analysis showed variable adduct abundance in many peptides between livers, but no concomitant decrease of unadducted peptides. This study sets a new standard for adduct detection in complex samples, offering insights into the human adductome resulting from reactive metabolite exposure. The methodology presented will aid mechanistic studies to identify, quantify and differentiate between adducts that result in adverse drug reactions and those that are benign.

Orthogonal separations of data from high-resolution mass spectrometry can provide insight into sample composition and address challenges of complete annotation of molecules in untargeted metabolomics. "Molecular networks" (MNs), as used in the Global Natural Products Social Molecular Networking platform, are a prominent strategy for exploring and visualizing molecular relationships and improving annotation. MNs are mathematical graphs showing the relationships between measured multidimensional data features. MNs also show promise for using network science algorithms to automatically identify targets for annotation candidates and to dereplicate features associated with a single molecular identity. This paper introduces "molecular hypernetworks" (MHNs) as more complex MN models able to natively represent multiway relationships among observations. Compared to MNs, MHNs can more parsimoniously represent the inherent complexity present among groups of observations, initially supporting improved exploratory data analysis and visualization. MHNs also promise to increase confidence in annotation propagation, for both human and analytical processing. We first illustrate MHNs with simple examples, and build them from liquid chromatography- and ion mobility spectrometry-separated MS data. We then describe a method to construct MHNs directly from existing MNs as their "clique reconstructions", demonstrating their utility by comparing examples of previously published graph-based MNs to their respective MHNs.

Lippia citriodora and Olea europaea are known for their shared common bioactivities. Although both matrices are rich in similar families of bioactive compounds, their specific phytochemical compounds are mostly different. Since these compounds can be metabolized in the organism, this study hypothesized that common bioavailable metabolites may contribute to their similar bioactive effects. To test this, an acute double-blind intervention study in humans was conducted with blood samples collected at multiple time points. Using an untargeted metabolomic approach based on HPLC-ESI-QTOF-MS, 66 circulating metabolites were detected, including 9 common to both extracts, such as homovanillic acid sulfate and glucuronide derivates, hydroxytyrosol sulfate, etc. These common metabolites displayed significantly different T max values depending on the source, suggesting distinct metabolization pathways for each extract. The study highlights how shared bioavailable metabolites may underlie similar bioactivities observed between these two plant sources.

Recent advancements in single-cell (sc) resolution analyses, particularly in sc transcriptomics and sc proteomics, have revolutionized our ability to probe and understand cellular heterogeneity. The study of metabolism through small molecules, metabolomics, provides an additional level of information otherwise unattainable by transcriptomics or proteomics by shedding light on the metabolic pathways that translate gene expression into functional outcomes. Metabolic heterogeneity, critical in health and disease, impacts developmental outcomes, disease progression, and treatment responses. However, dedicated approaches probing the sc metabolome have not reached the maturity of other sc omics technologies. Over the past decade, innovations in sc metabolomics have addressed some of the practical limitations, including cell isolation, signal sensitivity, and throughput. To fully exploit their potential in biological research, however, remaining challenges must be thoroughly addressed. Additionally, integrating sc metabolomics with orthogonal sc techniques will be required to validate relevant results and gain systems-level understanding. This perspective offers a broad-stroke overview of recent mass spectrometry (MS)-based sc metabolomics advancements, focusing on ongoing challenges from a biologist's viewpoint, aimed at addressing pertinent and innovative biological questions. Additionally, we emphasize the use of orthogonal approaches and showcase biological systems that these sophisticated methodologies are apt to explore.

Native mass spectrometry (MS) reveals the role of specific lipids in modulating membrane protein structure and function. Membrane proteins solubilized in detergents are often introduced into the mass spectrometer. However, detergents commonly used for structural studies, such as dodecylmaltoside, tend to generate highly charged ions, leading to protein unfolding, thereby diminishing their utility in characterizing protein-lipid interactions. Thus, there is a critical need to develop approaches to investigate protein-lipid interactions in different detergents. Here, we demonstrate how charge-reducing molecules, such as spermine and trimethylamine- N -oxide, enable the opportunity to characterize lipid binding to the bacterial water channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory protein GlnK in different detergent environments. We find that protein-lipid interactions not only are protein-dependent but also can be influenced by the detergent and type of charge-reducing molecule. AqpZ-lipid interactions are enhanced in LDAO ( n -dodecyl- N , N -dimethylamine- N -oxide), whereas the interaction of AmtB-GlnK with lipids is comparable among different detergents. A fluorescent lipid binding assay also shows detergent dependence for AqpZ-lipid interactions, consistent with results from native MS. Taken together, native MS will play a pivotal role in establishing optimal experimental parameters that will be invaluable for various applications, such as drug discovery as well as biochemical and structural investigations.

Coated blade spray-mass spectrometry (CBS-MS) has emerged as a powerful tool for the rapid screening of target compounds at trace levels in complex biological matrices. Despite its potential, the broader adoption of CBS-MS technology has been hindered by the lack of commercially available, user-friendly MS interfaces and extraction devices. In this work, we present comprehensive CBS-MS solutions developed using 3D printing, including a versatile MS interface and two extraction devices tailored to different analytical needs: one is compatible with LC vials for large-volume samples, while the other is optimized for single-drop blood analysis. The MS interface features a novel design that separates the immobilization station from the blade holder, significantly simplifying the operational workflow and minimizing the contamination risks and hazards associated with manual blade handling. The first extraction cartridge can process 48 samples simultaneously with an average sample preparation time of less than 20 s, while the second extraction device enables extraction from 8 single-drop blood samples via on-blade extraction. The developed devices were successfully tested for the rapid screening of seven drugs in both urine and single-drop blood samples, demonstrating a promising analytical performance. Additionally, potential contamination issues related to the use of 3D-printed materials as extraction phases were examined, emphasizing the importance of ensuring that 3D-printed materials do not leach contaminants into samples or solvents and contaminate the MS.

Current developments in single-cell mass spectrometry (MS) aim to deepen proteome coverage while enhancing analytical speed to study entire cell populations, one cell at a time. Custom-built microanalytical capillary electrophoresis (μCE) played a critical role in the foundation of discovery single-cell MS proteomics. However, requirements for manual operation, substantial expertise, and low measurement throughput have so far hindered μCE-based single-cell studies on large numbers of cells. Here, we design and construct a robotic capillary (RoboCap) platform that grants single-cell CE-MS with automation for proteomes limited to less than ∼100 nL. RoboCap remotely controls precision actuators to translate the sample to the fused silica separation capillary, using vials in this work. The platform is hermetically enclosed and actively pressurized to inject ∼1-250 nL of the sample into a CE separation capillary, with errors below ∼5% relative standard deviation (RSD). The platform and supporting equipment were operated and monitored remotely on a custom-written Virtual Instrument (LabView). Detection performance was validated empirically on ∼5-250 nL portions of the HeLa proteome digest using a trapped ion mobility mass spectrometer (timsTOF PRO). RoboCap improved CE-ESI sample utilization to ∼20% from ∼3% on the manual μCE, the closest reference technology. Proof-of-principle experiments found proteome identification and quantification to robustly return ∼1,800 proteins (∼13% RSD) from ∼20 ng of the HeLa proteome digest on this earlier-generation detector. RoboCap automates CE-MS for limited sample amounts, paving the way to electrophoresis-based high-throughput single-cell proteomics.

Protein-selection mass spectrometry is cost-effective for the discovery of drugs and toxics. Nuclear receptors (NRs) are major targets for pharmaceuticals and endocrine-disrupting chemicals and are, thus, widely used as "bait" proteins. However, their application is limited due to the tendency to lose protein activity during cold storage. To address this problem, we introduced a novel biomineralization-based approach to preserve activity in NRs, exemplified by human retinoic acid receptor alpha (hRARα), a target for cancer and leucocythemia therapy. Since information on the coordination chemistry of metal ion and NR protein complexes is almost unavailable, we applied peptide mapping analysis for the first time for the rational design of his-hRARα-Co phosphate nanobiomaterial with high bioactivity. This nanobiomaterial successfully captured hRARα bioactive chemicals from a Chinese herb and environmental water and discovered an unsaturated fatty acid, (±)-(9 Z ,11 E )-13-hydroxy-9,11-octadecadienoic acid ((±)13-HODE), which exhibited strong hRARα antagonistic activity.

Host cell proteins (HCPs) are contaminants of biotherapeutics produced from engineered living systems; they can influence the product's quality, efficacy, and toxicity. Liquid chromatography coupled to mass spectrometry can detect HCPs thereby mitigating their risks. However, highly abundant biotherapeutics hamper the detection of low-level HCPs. Sample preparation termed native digestion has proven effective to preferentially digest and draw out HCPs from intact antibodies. Here, we adapted native digestion to adeno-associated viruses (AAV), which is a vector gaining popularity for gene therapy. We leveraged quantitative proteomics using capillary-flow liquid chromatography-mass spectrometry (LC-MS) and demonstrated that native digestion was more effective than applying denaturing conditions to extract the HCPs associated with different AAV serotypes.

The uptake, translocation, and transformation of engineered nanoparticles (ENPs) in plants present significant challenges due to the lack of effective determination methods. This is especially true for selenium nanoparticles (SeNPs), which hold promise for Se-biofortified agriculture and exhibit dynamic behaviors within plant system. Herein, we proposed a novel approach that incorporates enzymic digestion and membrane filtration to selectively extract SeNPs and dissolved Se from plant tissues, employing rice ( Oryza sativa ) plant as a model. Subsequently, the SeNPs retained on the membrane were quantified using inductively coupled plasma mass spectrometry (ICPMS), while the dissolved Se in the filtrate, including selenite (Se(IV)), selenate (Se(VI)), and seleno amino acid, were analyzed by liquid chromatography coupled with ICPMS (LC-ICPMS). Recoveries of 83.5-91.4% for SeNPs and 73.6-99.4% for dissolved Se at a spiking level of 8 μg/g in quality control samples were obtained. With the established method, it was discovered that SeNPs taken up by rice leaves can transform into Se (IV) and organic Se, and all the Se species could be translocated downward, but only Se (IV) and SeNPs could be excreted through the roots. These findings provide valuable insights into the fate of SeNPs in plants and their related biological responses.

ortho -Phthalaldehyde (OPA) with high reactivity to the amine group has been widely used to modify proteins. We discovered new modifications of OPA and 2-acylbenzaldehyde and proposed the reaction mechanism. Using isotope labeling mass spectrometry-based experiment, we identified new cross-linking properties of OPA and 2-acylbenzaldehyde. This reactivity revealed that OPA has the potential to probe proximal amino acids in biological systems.