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Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (<1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

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Fucosylated carbohydrate antigens play critical roles in physiology and pathology with function linked to their structural details. However, the separation and structural characterization of isomeric fucosylated epitopes remain challenging analytically. Here, we report for the first time the influence of alkali metal cations (Li+, Na+, K+, Rb+, and Cs+) and halogen anions (Cl-, Br-, and I-) on the gas-phase conformational landscapes of common fucosylated trisaccharides (Lewis A, X, and H types 1 and 2) and tetrasaccharides (Lewis B and Y) using trapped ion mobility spectrometry coupled to mass spectrometry and theoretical calculations. Inspection of the mobility profiles of individual standards showed a dependence on the number of mobility bands with the oligosaccharide and the alkali metal and halogen; collision cross sections are reported for all of the observed species. Results showed that trisaccharides (Lewis A, X, and H types 1 and 2) can be best mobility resolved in the positive mode using the [M + Li]+ molecular ion form (baseline resolution r ≈ 2.88 between Lewis X and A); tetrasaccharides can be best mobility resolved in the negative mode using the [M + I]- molecular ion form (baseline separation r ≈ 1.35 between Lewis B and Y). The correlation between the number of oligosaccharide conformers as a function of the molecular ion adduct was studied using density functional theory. Theoretical calculations revealed that smaller cations can form more stable structures based on the number of coordinations, while larger cations induced greater oligosaccharide reorganizations; candidate structures are proposed to better understand the gas-phase oligosaccharide rearrangement trends. Inspection of the candidate structures suggests that the interplay between ion size/charge density and molecular structure dictated the conformational preferences and, consequently, the number of mobility bands and the mobility separation across isomers. This work provides a fundamental understanding of the gas-phase structural dynamics of fucosylated oligosaccharides and their interaction with alkali metals and halogens.

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The molecular level characterization of complex mixtures remains an analytical challenge. We have shown that the integration of complementary, high-resolution, gas-phase separations allows for chemical formula level isomeric content description. In the current work, we revisited the current challenges associated with the analysis of dissolved organic matter using high-resolution trapped ion mobility separation (TIMS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In particular, we evaluated the separation capabilities provided by TIMS-MS compared to MS alone, the use of ICR complementary data acquisition (DAQ) systems and transient processing strategies, ICR cell geometries (e.g., Infinity cell vs harmonized cell), and magnetic field strengths (7 T vs 9.4 T vs 21 T) for the case of a Harney River DOM sample. Results showed that the external high-performance DAQ enables direct representation of mass spectra in absorption mode FT (aFT), doubling the MS resolution compared to the default magnitude mode FT (mFT). Changes between half- vs full-apodization result in greater MS signal/noise vs superior MS resolving power (RP); in the case of DOM analysis, a 45% increase in assigned formulas is observed when employing the DAQ half (Kaiser-type)-apodization window and aFT when compared to the default instrument mFT. Results showed the advantages of reprocessing 2D-TIMS-FT-ICR MS data with higher RP and magnetic field chemical formulas generated list acquired (e.g., 21 T led to a 24% increase in isomers reported) or the implementation of alternative strategies.

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The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K0) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me1-3) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.

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Solution-phase hydrogen/deuterium exchange (HDX) coupled to native ion mobility spectrometry mass spectrometry (IMS-MS) can provide complementary structural information about the conformational dynamics of biological molecules. In the present work, the solution-stable isotope labeling (SIL) combined with trapped ion mobility spectrometry (TIMS) in tandem with top-down electron capture dissociation (ECD) is illustrated for the structural characterization of the solution native states of ubiquitin. Four different ubiquitin electrospray solution conditions: (i) single-tip nondeuterated, (ii) theta tip for online SIL HDX, (iii) single-tip SIL-deuterated, and (iv) theta tip for online SIL H/D back exchange (HDbX), were investigated to assess the H/D exchange reactivities of native ubiquitin. The combination of TIMS and ECD in a q-ToF MS instrument allowed for additional inspection of gas-phase HDbX added by top-down fragmentation, revealing the exposed and protected residues with limited scrambling effects (e.g., intramolecular H/D migration). A native charge state distribution (5+ to 7+) and TIMS profiles were observed under the single-tip nondeuterated solution conditions. Mass shift distributions of ∼40, ∼104, and ∼87D were observed when incorporating deuterium for online SIL HDX, SIL HDX, and online SIL HDbX, respectively, while retaining similar conformational states. ECD fragmentation allowed for the localization of the deuterated labeled residues of the peptide fragments, with a sequence coverage of ∼90%, for each of the ubiquitin solution condition. Changes in the TIMS trapping time settings (∼70 to ∼795 ms) were used to determine the H/D back exchange dynamics of native ubiquitin. HDbX-TIMS-q-ECD-MS/MS exhibited H/D back exchanges in the six-residue C-terminal tail as well as around Lys6, Lys11, Lys33, Lys48, and Lys63 residues, indicating that these regions are the most exposed area (less protected hydrogens) of ubiquitin as compared to the rest of the core residues that adopt a compact ß-grasp fold (protected hydrogens), which was consistent with the accessible surface area of ubiquitin. The present data highlight for the first time consistency between the solution HDX and gas-phase HDbX-TIMS data for native studies.

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Knotted proteins are fascinating natural biomolecules whose backbones entangle themselves in a knot. Their particular knotted configurations provide them with a wide range of topological features. However, their folding/unfolding mechanisms, stability, and function are poorly understood. In the present work, native trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used for characterizing structural features of two model knotted proteins: a Gordian 52 knot ubiquitin C-terminal hydrolase (UCH) and a Stevedore 61 knot (α-haloacid dehalogenase, DehI). Experimental results showed structural transitions of UCH and DehI as a function of solution composition (0-50% MeOH) and temperature (T ∼20-95 °C). An increase in the protein charge states and collision cross sections (∼2750-8750 Å2 and ∼3250-15,385 Å2 for UCH and DehI, respectively) with the solution organic content (OC) and temperature suggested a three-step unfolding pathway with at least four structural transitions. Results also showed that the integrity of the UCH knot core was more resistant to thermal unfolding when compared to DehI; however, both knot cores can be disrupted with the increase in the solution OC. Additional enzymatic digestion experiments using carboxypeptidase Y combined with molecular dynamics simulations showed that the knot core was preserved between Glu20 and Glu188 and Arg89 and His304 residues for UCH and DehI, respectively, where disruption of the knot core led to structural collapse followed by unfolding events. This work highlights the potential of solution OC and temperature studies combined with native TIMS-MS for the comprehensive characterization of knotted proteins to gain a better understanding of their structural transitions.

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Daunorubicin and doxorubicin are among the most potent anti-cancer drugs and bind to DNA through intercalation. In this paper, we demonstrate that formaldehyde can efficiently and specifically conjugate daunorubicin and doxorubicin to GTP, resulting in the formation of daunorubicin-GTP-1 and doxorubicin-GTP-1 conjugates. The linkage occurs between the 2-NH2 of guanine and the 3'-NH2 of daunosamine. We characterized these daunorubicin/doxorubicin-GTP conjugates using various methods, including UV-Vis, fluorescence, CD, FT-IR, and mass spectrometry. Our results also indicate that these daunorubicin/doxorubicin-GTP conjugates bind to DNA via intercalation. Furthermore, we observed rapid accumulation of these conjugates in human cancer cells and observed cytotoxic effects in both doxorubicin-sensitive SK-OV-3 and doxorubicin-resistant NCI/ADR-RES cells, suggesting that these daunorubicin and doxorubicin derivatives can overcome doxorubicin resistance.

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Known for their high stability and surfactant properties, per- and polyfluoroalkyl substances (PFAS) have been widely used in a range of manufactured products. Despite being largely phased out due to concerns regarding their persistence, bioaccumulation, and toxicity, legacy PFAS such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid continue to persist at high levels in the environment, posing risks to aquatic organisms. We used high-resolution magic angle spinning nuclear magnetic resonance spectroscopy in intact zebrafish (Danio rerio) embryos to investigate the metabolic pathways altered by PFOS both before and after hatching (i.e., 24 and 72 h post fertilization [hpf], respectively). Assessment of embryotoxicity found embryo lethality in the parts-per-million range with no significant difference in mortality between the 24- and 72-hpf exposure groups. Metabolic profiling revealed mostly consistent changes between the two exposure groups, with altered metabolites generally associated with oxidative stress, lipid metabolism, energy production, and mitochondrial function, as well as specific targeting of the liver and central nervous system as key systems. These metabolic changes were further supported by analyses of tissue-specific production of reactive oxygen species, as well as nontargeted mass spectrometric lipid profiling. Our findings suggest that PFOS-induced metabolic changes in zebrafish embryos may be mediated through previously described interactions with regulatory and transcription factors leading to disruption of mitochondrial function and energy metabolism. The present study proposes a systems-level model of PFOS toxicity in early life stages of zebrafish, and also identifies potential biomarkers of effect and exposure for improved environmental biomonitoring. Environ Toxicol Chem 2024;43:896-914. © 2024 SETAC.

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Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

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α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

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Opiate abuse increases the risk of HIV transmission and exacerbates HIV neuropathology by increasing inflammation and modulating immune cell function. Exosomal EVs(xEV) contain miRNAs that may be differentially expressed due to HIV infection or opiate abuse. Here we develop a preliminary exosomal-miRNA biomarker profile of HIV-infected PBMCs in the context of opiate use. PBMCs infected with HIV were treated with increasing dosages of morphine for 72 hours, the culture supernatants were collected, and the exosomes isolated using differential centrifugation. Exosomal miRNAs were extracted, expression levels determined via Nanostring multiplexed microRNA arrays, and analyzed with Webgestalt. The effect of the exosomes on neuronal function was determined by measuring calcium. Preliminary findings show that HIV-1 infection altered the miRNA profile of PBMC-derived EVs concurrently with opiate exposure. MicroRNA, hsa-miR-1246 was up-regulated 12-fold in the presence of morphine, relative to uninfected control. PBMCs infected with HIV-1 MN, an X4-tropic HIV-1 strain and exposed to morphine, displayed a trend which suggests potential synergistic effects between HIV-1 infection and morphine exposure promoting an increase in viral replication. Dose-dependent differences were observed in miRNA expression as a result of opiate exposure. The xEVs derived from PBMCs exposed to morphine or HIV modulated neuronal cell function. SH-SY5Y cells, treated with xEVs derived from ART-treated PBMCs, exhibited increased viability while for SH-SY5Ys exposed to xEVs derived from HIV-1 infected PBMCs viability was decreased compared to the untreated control. Exposing SH-SY5Y to xEVs derived from HIV-infected PBMCs resulted in significant decrease in calcium signaling, relative to treatment with xEVs derived from uninfected PBMCs. Overall, HIV-1 and morphine induced differential miRNA expression in PBMC-derived exosomes, potentially identifying mechanisms of action or novel therapeutic targets involved in opiate use disorder, HIV neuropathology, TNF signaling pathway, NF-κB signaling pathway, autophagy, and apoptosis in context of HIV infection.

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The blood-sucking hemipteran Rhodnius prolixus is one of the main vectors of Chagas disease, a neglected tropical disease that affects several million people worldwide. Consuming a blood meal and mating are events with a high epidemiological impact since after each meal, mated females can lay fertile eggs that result in hundreds of offspring. Thus, a better knowledge of the control of R. prolixus reproductive capacity may provide targets for developing novel strategies to control vector populations, thereby reducing vector-host contacts and disease transmission. Here, we have used a combination of gene transcript expression analysis, biochemical assays, hormone measurements and studies of locomotory activity to investigate how mating influences egg development and egg laying rates in R. prolixus females. The results demonstrate that a blood meal increases egg production capacity and leads to earlier egg laying in mated females compared to virgins. Virgin females, however, have increased survival rate over mated females. Circulating juvenile hormone (JH) and ecdysteroid titers are increased in mated females, a process mainly driven through an upregulation of the transcripts for their biosynthetic enzymes in the corpus allatum and ovaries, respectively. Mated females display weaker locomotory activity compared to virgin females, mainly during the photophase. In essence, this study shows how reproductive output and behaviour are profoundly influenced by mating, highlighting molecular, biochemical, endocrine and behavioral features differentially expressed in mated and virgin R. prolixus females.

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There is a growing demand for lower-cost, benchtop analytical instruments with complementary separation capabilities for the screening and characterization of biological samples. In this study, we report on the custom integration of trapped ion mobility spectrometry and ultraviolet photodissociation capabilities in a commercial Paul quadrupolar ion trap multistage mass spectrometer (TIMS-QIT-MSn UVPD platform). A gated TIMS operation allowed for the accumulation of ion mobility separated ion in the QIT, followed by a mass analysis (MS1 scan) or m/z isolation, followed by selected collision induced dissociation (CID) or ultraviolet photodissociation (UVPD) and a mass analysis (MS2 scan). The analytical potential of this platform for the analysis of complex and labile biological samples is illustrated for the case of positional isomers with varying PTM location of the histone H4 tryptic peptide 4-17 singly and doubly acetylated and the histone H3.1 tail (1-50) singly trimethylated. For all cases, a baseline ion mobility precursor molecular ion preseparation was obtained. The tandem CID and UVPD MS2 allowed for effective sequence confirmation as well as the identification of reporter fragment ions associated with the PTM location; a higher sequence coverage was obtained using UVPD when compared to CID. Different from previous IMS-MS implementation, the novel TIMS-QIT-MSn UVPD platform offers a lower-cost alternative for the structural characterization of biological molecules that can be widely disseminated in clinical laboratories.

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Lanthanides have been frequently used as biomimetic compounds for NMR and fluorescence studies of Ca2+ binding proteins due to having similar physical properties and coordination geometry to Ca2+ ions. Here we report that a member of the neuronal calcium sensor family, neuronal calcium sensor 1, complexes with two lanthanide ions Tb3+ and Eu3+. The affinity for Tb3+ is nearly 50 times higher than that for Ca2+ (Kd,Tb3+ = 0.002 ± 0.0001 µM and Kd, Ca2+ = 91 nM) whereas Eu3+ binding is notably weaker, Kd,Eu3+ = 26 ± 1 µM. Interestingly, despite having identical charge and similar ionic radii, Tb3+ and Eu3+ ions exhibit a distinct binding stoichiometry for NCS1 with one Eu3+ and two Tb3+ ions bound per NCS1 monomer, as demonstrated in fluorescence titration and mass spectrometry studies. These results suggest that the lanthanides' affinity for the individual EF hands is fine-tuned by a small variation in the ion charge density as well as EF hand binding loop amino acid sequence. As observed previously for other lanthanide:protein complexes, the emission intensity of Ln3+ is enhanced upon complexation with the protein, likely due to the displacement of water molecules by oxygen atoms from the coordinating amino acid residues. The overall shape of the Tb3+NCS1 and Eu3+NCS1 monomer shows high levels of similarity compared to the Ca2+ bound protein based on their collision cross section. However, the distinct occupation of EF hands impacts NCS1 oligomerization and affinity for the D2R peptide that mimics the NCS1 binding site on the D2R receptor. Specifically, the Tb3+NCS1 complex populates the dimer and has comparable affinity for the D2R peptide, whereas Eu3+ bound NCS1 remains in the monomeric form with a negligible affinity for the D2R peptide.

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Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant (Kd) in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms.

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Dissolved Organic Matter (DOM) is an important component of the global carbon cycle. Unscrambling the structural footprint of DOM is key to understand its biogeochemical transformations at the mechanistic level. Although numerous studies have improved our knowledge of DOM chemical makeup, its three-dimensional picture remains largely unrevealed. In this work, we compare four solid phase extracted (SPE) DOM samples from three different freshwater ecosystems using high resolution mobility and ultrahigh-resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FT-ICR MS/MS). Structural families were identified based on neutral losses at the level of nominal mass using continuous accumulation of selected ions-collision induced dissociation (CASI-CID)FT-ICR MS/MS. Comparison of the structural families indicated dissimilarities in the structural footprint of this sample set. The structural family representation using Cytoscape software revealed characteristic clustering patterns among the DOM samples, thus confirming clear differences at the structural level (Only 10% is common across the four samples.). The analysis at the level of neutral loss-based functionalities suggests that hydration and carboxylation are ubiquitous transformational processes across the three ecosystems. In contrast, transformation mechanisms involving methoxy moieties may be constrained in estuarine systems due to extensive upstream lignin biodegradation. The inclusion of the isomeric content (mobility measurements at the level of chemical formula) in the structural family description suggests that additional transformation pathways and/or source variations are possible and account for the dissimilarities observed. While the structural character of more and diverse types of DOM samples needs to be assessed and added to this database, the results presented here demonstrate that Graph-DOM is a powerful tool capable of providing novel information on the DOM chemical footprint, based on structural interconnections of precursor molecules generated by fragmentation pathways and collisional cross sections.

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Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

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Juvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. Therefore, achieving tissue-specific expression of transgenes in the CA would be beneficial for mosquito research and control. Different CA promoters have been used to drive transgene expression in Drosophila, but mosquito CA-specific promoters have not been identified. Using the CRISPR/Cas9 system, we integrated transgenes encoding the reporter green fluorescent protein (GFP) close to the transcription start site of juvenile hormone acid methyl transferase (JHAMT), a locus encoding a JH biosynthetic enzyme, specifically and highly expressed in the CA of Aedes aegypti mosquitoes. Transgenic individuals showed specific GFP expression in the CA but failed to reproduce the full pattern of jhamt spatiotemporal expression. In addition, we created GeneSwitch driver and responder mosquito lines expressing an inducible fluorescent marker, enabling the temporal regulation of the transgene via the presence or absence of an inducer drug. The use of the GeneSwitch system has not previously been reported in mosquitoes and provides a new inducible binary system that can control transgene expression in Aedes aegypti.

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Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approach─ultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)─is illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (∼75%) with significant faster data acquisition in the ToF MS/MS platform (∼3 vs ∼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (∼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c305+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me2, 2Ac + Me2 and 3Ac + Me2), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c305+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.

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