Offline Two-Dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome Following Fecal Microbiota Transplantation.

Biological interpretation of untargeted LC-MS-based metabolomics data depends on accurate compound identification, but current techniques fall short of identifying most features that can be detected. The human fecal metabolome is complex, variable, incompletely annotated, and serves as an ideal matrix to evaluate novel compound identification methods. We devised an experimental strategy for compound annotation using multidimensional chromatography and semiautomated feature alignment and applied these methods to study the fecal metabolome in the context of fecal microbiota transplantation (FMT) for recurrent C. difficile infection. Pooled fecal samples were fractionated using semipreparative liquid chromatography and analyzed by an orthogonal LC-MS/MS method. The resulting spectra were searched against commercial, public, and local spectral libraries, and annotations were vetted using retention time alignment and prediction. Multidimensional chromatography yielded more than a 2-fold improvement in identified compounds compared to conventional LC-MS/MS and successfully identified several rare and previously unreported compounds, including novel fatty-acid conjugated bile acid species. Using an automated software-based feature alignment strategy, most metabolites identified by the new approach could be matched to features that were detected but not identified in single-dimensional LC-MS/MS data. Overall, our approach represents a powerful strategy to enhance compound identification and biological insight from untargeted metabolomics data.

Neurochemical and Neurophysiological Effects of Intravenous Administration of N,N-dimethyltryptamine in Rats.

N,N-dimethyltryptamine (DMT) is a serotonergic psychedelic that is being investigated clinically for the treatment of psychiatric disorders. Although the neurophysiological effects of DMT in humans are well-characterized, similar studies in animal models as well as data on the neurochemical effects of DMT are generally lacking, which are critical for mechanistic understanding. In the current study, we combined behavioral analysis, high-density (32-channel) electroencephalography, and ultra-high-performance liquid chromatography-tandem mass spectrometry to simultaneously quantify changes in behavior, cortical neural dynamics, and levels of 17 neurochemicals in medial prefrontal and somatosensory cortices before, during, and after intravenous administration of three different doses of DMT (0.75 mg/kg, 3.75 mg/kg, 7.5 mg/kg) in male and female adult rats. All three doses of DMT produced head twitch response with most twitches observed after the low dose. DMT caused dose-dependent increases in serotonin and dopamine levels in both cortical sites along with a reduction in EEG spectral power in theta (4-10 Hz) and low gamma (25-55 Hz), and increase in power in delta (1-4 Hz), medium gamma (65-115), and high gamma (125-155 Hz) bands. Functional connectivity decreased in the delta band and increased across the gamma bands. In addition, we provide the first measurements of endogenous DMT in these cortical sites at levels comparable to serotonin and dopamine, which together with a previous study in occipital cortex, suggests a physiological role for endogenous DMT. This study represents one of the most comprehensive characterizations of psychedelic drug action in rats and the first to be conducted with DMT.

Systematic evaluation of benzoylation for liquid chromatography-mass spectrometry analysis of different analyte classes.

LC-MS is an indispensable tool for small molecule analysis in many fields; however, many small molecules require chemical derivatization to improve retention on commonly used reversed-phase columns and increase ionization. Benzoyl chloride (BzCl) derivatization is commonly used for derivatization of primary and secondary amines and phenolic alcohols, though evidence exists that with proper reaction conditions (i.e., specific bases), other hydroxyl groups may be derivatized too. Previous studies have examined BzCl concentration, reaction times, and reaction temperatures for derivatization of amines and phenols for LC-MS analysis; however, use of different bases, base concentration, and extending to conditions to hydroxyl groups for LC-MS analysis has not been well-studied. To address this understudied area and identify reaction conditions for both amino and hydroxyl groups, we performed a systematic study of reaction conditions on multiple classes of potential targets. For selected derivatization methods, detection limits and performance in a variety of biological matrices were assessed. Results highlight the importance of tailoring derivatization methods for a given application as they varied by molecule and/or molecule class. Compared to the standard BzCl method commonly used, alternative methods were identified to better derivatize challenging analytes (glucosamine, choline, cortisol, uridine, cytidine) with detection limits reaching 1100, 9, 38, 170, and 67 nM compared to undetectable, 170, 86, 1000, and 86 nM respectively. Sub-nanomolar detection limits were achieved for norepinephrine with alternative derivatization approaches. Improved derivatization methods for several classes and molecules including nucleosides, steroids, and molecules containing hydroxyl groups were also identified.

Preparation of high-efficiency HILIC capillary columns utilizing slurry packing at 2100 bar.

Complex mixture analysis requires high-efficiency chromatography columns. Although reversed phase liquid chromatography (RPLC) is the dominant approach for such mixtures, hydrophilic interaction liquid chromatography (HILIC) is an important complement to RPLC by enabling the separation of polar compounds. Chromatography theory predicts that small particles and long columns will yield high efficiency; however, little work has been done to prepare HILIC columns longer than 25 cm packed with sub-2 µm particles. In this work, we tested the slurry packing of 75 cm long HILIC columns with 1.7 µm bridged-ethyl-hybrid amide HILIC particles at 2,100 bar (30,000 PSI). Acetonitrile, methanol, acetone, and water were tested as slurry solvents, with acetonitrile providing the best columns. Slurry concentrations of 50-200 mg/mL were assessed, and while 50-150 mg/mL provided comparable results, the 150 mg/mL columns provided the shortest packing times (9 min). Columns prepared using 150 mg/mL slurries in acetonitrile yielded a reduced minimum plate height (hmin) of 3.3 and an efficiency of 120,000 theoretical plates for acenaphthene, an unretained solute. Para-toluenesulfonic acid produced the lowest hmin of 1.9 and the highest efficiency of 210,000 theoretical plates. These results identify conditions for producing high-efficiency HILIC columns with potential applications to complex mixture analysis.

Cyclic Ion Mobility-Mass Spectrometry and Tandem Collision Induced Unfolding for Quantification of Elusive Protein Biomarkers.

Sensitive analytical techniques that are capable of detecting and quantifying disease-associated biomolecules are indispensable in our efforts to understand disease mechanisms and guide therapeutic intervention through early detection, accurate diagnosis, and effective monitoring of disease. Parkinson's Disease (PD), for example, is one of the most prominent neurodegenerative disorders in the world, but the diagnosis of PD has primarily been based on the observation of clinical symptoms. The protein α-synuclein (α-syn) has emerged as a promising biomarker candidate for PD, but a lack of analytical methods to measure complex disease-associated variants of α-syn has prevented its widespread use as a biomarker. Antibody-based methods such as immunoassays and mass spectrometry-based approaches have been used to measure a limited number of α-syn forms; however, these methods fail to differentiate variants of α-syn that display subtle differences in only the sequence and structure. In this work, we developed a cyclic ion mobility-mass spectrometry method that combines multiple stages of activation and timed ion selection to quantify α-syn variants using both mass- and structure-based measurements. This method can allow for the quantification of several α-syn variants present at physiological levels in biological fluid. Taken together, this approach can be used to galvanize future efforts aimed at understanding the underlying mechanisms of PD and serves as a starting point for the development of future protein-structure-based diagnostics and therapeutic interventions.

High-Throughput Capillary Liquid Chromatography Using a Droplet Injection and Application to Reaction Screening.

The cycle time of a standard liquid chromatography (LC) system is the sum of the time for the chromatographic run and the autosampler injection sequence. Although LC separation times in the 1-10 s range have been demonstrated, injection sequences are commonly >15 s, limiting throughput possible with LC separations. Further, such separations are performed on relatively large bore columns requiring flow rates of ≥5 mL/min, thus generating large volumes of mobile phase waste when used for large scale screening and increasing the difficulty in interfacing to mass spectrometry. Here, a droplet injector system was established that replaces the autosampler with a four-port, two-position valve equipped with a 20 nL internal loop interfaced to a syringe pump and a three-axis positioner to withdraw sample droplets from a well plate. In the system, sample and immiscible fluid are pulled alternately from a well plate into a capillary and then through the injection valve. The valve is actuated when sample fills the loop to allow sequential injection of samples at high throughput. Capillary LC columns with 300 µm inner diameter were used to reduce the consumption of mobile phase and sample. The system achieved 96 separations of 20 nL droplet samples containing 3 components in as little as 8.1 min with 5-s cycle time. This system was coupled to a mass spectrometer through an electrospray ionization source for high-throughput chemical reaction screening.

Microdialysis coupled with droplet microfluidics and mass spectrometry for determination of neurotransmitters in vivo with high temporal resolution.

Monitoring the concentration fluctuations of neurotransmitters in vivo is valuable for elucidating the chemical signals that underlie brain functions. Microdialysis sampling is a widely used tool for monitoring neurochemicals in vivo. The volume requirements of most techniques that have been coupled to microdialysis, such as HPLC, result in fraction collection times of minutes, thus limiting the temporal resolution possible. Further the time of analysis can become long for cases where many fractions are collected. Previously we have used direct analysis of dialysate by low-flow electrospray ionization-tandem mass spectrometry (ESI-MS/MS) on a triple quadrupole mass spectrometer to monitor acetylcholine, glutamate, and γ-amino-butyric acid to achieve multiplexed in vivo monitoring with temporal resolution of seconds. Here, we have expanded this approach to adenosine, dopamine, and serotonin. The method achieved limits of detection down to 2 nM, enabling basal concentrations of all these compounds, except serotonin, to be measured in vivo. Comparative analysis with LC-MS/MS showed accurate results for all compounds except for glutamate, possibly due to interference for this compound in vivo. Pairing this analysis with droplet microfluidics yields 11 s temporal resolution and can generate dialysate fractions down to 3 nL at rates up to 3 fractions per s from a microdialysis probe. The system is applied to multiplexed monitoring of neurotransmitter dynamics in response to stimulation by 100 mM K+ and amphetamine. These applications demonstrate the suitability of the droplet ESI-MS/MS method for monitoring short-term dynamics of up to six neurotransmitters simultaneously.

Expedited loading with intravenous sotalol is safe and feasible-primary results of the Prospective Evaluation Analysis and Kinetics of IV Sotalol (PEAKS) Registry.

BACKGROUND: Loading of oral sotalol for atrial fibrillation requires 3 days, frequently in the hospital, to achieve steady state. The Food and Drug Administration approved loading with intravenous (IV) sotalol through model-informed development, without patient data. OBJECTIVE: We present results of the first multicenter evaluation of this recent labeling for IV sotalol. METHODS: The Prospective Evaluation Analysis and Kinetics of IV Sotalol (PEAKS) Registry was a multicenter observational registry of patients undergoing elective IV sotalol load for atrial arrhythmias. Outcomes, measured from hospital admission until first outpatient follow-up, included adverse arrhythmia events, efficacy, and length of stay. RESULTS: Of 167 consecutively enrolled patients, 23% were female; the median age was 68 (interquartile range, 61-74) years, and the median CHA2DS2-VASc score was 3 (interquartile range, 2-4). Overall, 99% were admitted for sotalol initiation (1% for dose escalation), with a target oral sotalol dose of either 80 mg twice daily (85 [51%]) or 120 mg twice daily (78 [47%]); 62 patients (37%) had an estimated creatinine clearance ≤90 mL/min. On presentation, 40% of patients were in sinus rhythm, whereas 26% underwent cardioversion before sotalol infusion. In 2 patients, sotalol infusion was stopped for bradycardia or hypotension. In 6 patients, sotalol was discontinued before discharge because of QTc prolongation (3), bradycardia (1), or recurrent atrial arrhythmia (2). The mean length of stay was 1.1 days, and 95% (n = 159) were discharged within 1 night. CONCLUSION: IV sotalol loading is safe and feasible for atrial arrhythmias, with low rates of adverse events, and yields shorter hospitalizations. More data are needed on the minimal duration required for monitoring in the hospital.

Supported Platinum Nanoparticles Catalyzed Carbon-Carbon Bond Cleavage of Polyolefins: Role of the Oxide Support Acidity.

Supported platinum nanoparticle catalysts are known to convert polyolefins to high-quality liquid hydrocarbons using hydrogen under relatively mild conditions. To date, few studies using platinum grafted onto various metal oxide (MxOy) supports have been undertaken to understand the role of the acidity of the oxide support in the carbon-carbon bond cleavage of polyethylene under consistent catalytic conditions. Specifically, two Pt/MxOy catalysts (MxOy = SrTiO3 and SiO2-Al2O3; Al = 3.0 wt %, target Pt loading 2 wt % Pt ∼1.5 nm), under identical catalytic polyethylene hydrogenolysis conditions (T = 300 °C, P(H2) = 170 psi, t = 24 h; Mw = ∼3,800 g/mol, Mn = ∼1,100 g/mol, D = 3.45, Nbranch/100C = 1.0), yielded a narrow distribution of hydrocarbons with molecular weights in the range of lubricants (Mw = < 600 g/mol; Mn < 400 g/mol; D = 1.5). While Pt/SrTiO3 formed saturated hydrocarbons with negligible branching, Pt/SiO2-Al2O3 formed partially unsaturated hydrocarbons (<1 mol % alkenes and ∼4 mol % alkyl aromatics) with increased branch density (Nbranch/100C = 5.5). Further investigations suggest evidence for a competitive hydrocracking mechanism occurring alongside hydrogenolysis, stemming from the increased acidity of Pt/SiO2-Al2O3 compared to Pt/SrTiO3. Additionally, the products of these polymer deconstruction reactions were found to be independent of the polyethylene feedstock, allowing the potential to upcycle polyethylenes with various properties into a value-added product.

High-Throughput Liquid Chromatographic Analysis Using a Segmented Flow Injector with a 1 s Cycle Time.

High-throughput screening (HTS) workflows are revolutionizing many fields, including drug discovery, reaction discovery and optimization, diagnostics, sensing, and enzyme engineering. Liquid chromatography (LC) is commonly deployed during HTS to reduce matrix effects, distinguish isomers, and preconcentrate prior to detection, but LC separation time often limits throughput. Although subsecond LC separations have been demonstrated, they are rarely utilized during HTS due to limitations associated with the speed of common autosamplers. In this work, these limits are overcome by utilizing droplet microfluidics for sample introduction. In the method, a train of samples segmented by air are continuously pumped into the inlet of an LC injection valve that is actuated once each sample fills the sample loop. Coupled with 2.1 mm diameter × 5 mm long columns packed with 2.7 µm superficially porous C18 particles operated at 5 mL/min, the injector enabled separation of 3 components at 1 s/sample and analysis of a 96-well plate in 1.6 min with <2% peak area relative standard deviation. Analyte-dependent carryover was minimized by including wash droplets composed of organic solvent in between sample droplets. High-throughput LC coupled with mass spectrometric detection using the segmented flow injector was applied to a screen of inhibitors of a cytochrome P450-catalyzed hydroxylation reaction. Measurements of the reaction substrate and product concentrations made using fast LC with the segmented flow injector correlated well with measurements made using a more conventional, 3 min LC method. These results demonstrate the potential for droplet microfluidics to be used for sample introduction during high-throughput LC analysis.

Conserved 5-methyluridine tRNA modification modulates ribosome translocation.

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m 5 U54). Here, we uncover contributions of m 5 U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m 5 U in the T-loop (TrmA in E. coli , Trm2 in S. cerevisiae ) exhibit altered tRNA modifications patterns. Furthermore, m 5 U54 deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that, relative to wild-type cells, trm2 Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m 5 U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

Evaluation of Surface Treatments of PDMS Microfluidic Devices for Improving Small-Molecule Recovery with Application to Monitoring Metabolites Secreted from Islets of Langerhans.

Microfluidic devices are becoming an important tool for bioanalysis with applications including studying cell secretion, cell growth, and drug delivery. Small molecules such as drugs, cell products, or nutrients may partition into polydimethylsiloxane (PDMS), a commonly used material for microfluidic devices, potentially leading to poor recovery or inaccurate delivery of such chemicals. To decrease small-molecule partitioning, surface and bulk PDMS treatments have been developed; however, these have been tested on few analytes, or their biocompatibility are unknown. Studies often focus on one analyte, whereas a diversity of chemicals are of interest and possibly affected. In this study, 11 device treatments are tested and applied to 21 biologically relevant small molecules with a variety of chemical structures. Device treatments are characterized using water contact angle measurements and evaluated by measuring recovery of the 21 target analytes using liquid chromatography-mass spectrometry. 1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene), a positively charged polymer, produced the least hydrophilic surface and was found to provide the best recovery with most of the analytes having >50% recovery and up to 92% recovery; however, recovery varied by analyte highlighting the importance of analyte diversity rather than targeting a single analyte in evaluating treatments. A polybrene-treated device was applied to investigate secretion from pancreatic islets, which are micro-organs involved in glucose homeostasis and diabetes. Islets secrete small molecules that have been shown to modulate the secretion of islets' main functional products, glucose-regulating hormones. The polybrene treatment enabled the detection of 20 target analytes from islets-on-chip during isosmotic and hypo-osmotic glucose perfusions and resulted in detection of more significant secretion changes compared to untreated PDMS.

Mass-Activated Droplet Sorting for the Selection of Lysine-Producing Escherichia coli.

Synthetic biology relies on engineering cells to have desirable properties, such as the production of select chemicals. A bottleneck in engineering methods is often the need to screen and sort variant libraries for potential activity. Droplet microfluidics is a method for high-throughput sample preparation and analysis which has the potential to improve the engineering of cells, but a limitation has been the reliance on fluorescent analysis. Here, we show the ability to select cell variants grown in 20 nL droplets at 0.5 samples/s using mass-activated droplet sorting (MADS), a method for selecting droplets based on the signal intensity measured by electrospray ionization mass spectrometry (ESI-MS). Escherichia coli variants producing lysine were used to evaluate the applicability of MADS for synthetic biology. E. coli were shown to be effectively grown in droplets, and the lysine produced by these cells was detectable using ESI-MS. Sorting of lysine-producing cells based on the MS signal was shown, yielding 96-98% purity for high-producing variants in the selected pool. Using this technique, cells were recovered after screening, enabling downstream validation via phenotyping. The presented method is translatable to whole-cell engineering for biocatalyst production.

Caspase activation in tumour-infiltrating lymphocytes is associated with lymph node metastasis in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCCs) are genetically heterogeneous and exhibit diverse stromal and immune microenvironments. Acquired resistance to standard chemo-, radio-, and targeted therapies remains a major hurdle in planning effective treatment modalities for OSCC patients. Since Caspase 8 (CASP8) is frequently mutated in OSCCs, we were interested to explore a potential interaction between tumour-infiltrating lymphocytes (TILs) and CASP8 activation using high-content image analysis of human tumour (n = 32) sections. Despite the lymphocyte-rich tumour microenvironment, we observed lower activation of CASP8 (0-10% of tumour area) and its downstream effector CASP3 (0-6%) in tumours than in normal oral epithelium. Conversely, we found apoptosis was high for all the lymphocyte subtypes examined (38-52% of lymphocytes within tumour islands). Tumours with higher Fas ligand (FasL) expression had a significantly higher proportion of cleaved CASP3/8 positive cytotoxic T cells within the tumour islands (p = 0.05), and this was associated with the presence of lymph node metastatic disease [odds ratio: 1.046, 95% confidence interval (1.002-1.091), p = 0.039]. Our finding of extensive activation of the extrinsic pathway of apoptosis in TILs, together with evidence of higher FasL in CASP8 mutated tumours, may be useful in predicting the course of disease in individual patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Offline Two-dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome.

Compound identification is an essential task in the workflow of untargeted metabolomics since the interpretation of the data in a biological context depends on the correct assignment of chemical identities to the features it contains. Current techniques fall short of identifying all or even most observable features in untargeted metabolomics data, even after rigorous data cleaning approaches to remove degenerate features are applied. Hence, new strategies are required to annotate the metabolome more deeply and accurately. The human fecal metabolome, which is the focus of substantial biomedical interest, is a more complex, more variable, yet lesser-investigated sample matrix compared to widely studied sample types like human plasma. This manuscript describes a novel experimental strategy using multidimensional chromatography to facilitate compound identification in untargeted metabolomics. Pooled fecal metabolite extract samples were fractionated using offline semi-preparative liquid chromatography. The resulting fractions were analyzed by an orthogonal LC-MS/MS method, and the data were searched against commercial, public, and local spectral libraries. Multidimensional chromatography yielded more than a 3-fold improvement in identified compounds compared to the typical single-dimensional LC-MS/MS approach and successfully identified several rare and novel compounds, including atypical conjugated bile acid species. Most features identified by the new approach could be matched to features that were detectable but not identifiable in the original single-dimension LC-MS data. Overall, our approach represents a powerful strategy for deeper annotation of the metabolome that can be implemented with commercially-available instrumentation, and should apply to any dataset requiring deeper annotation of the metabolome.

Advances in coupling droplet microfluidics to mass spectrometry.

Droplet microfluidics enables development of workflows with low sample consumption and high throughput. Fluorescence-based assays are most used with droplet microfluidics; however, the requirement of a fluorescent reporter restricts applicability of this approach. The coupling of droplets to mass spectrometry (MS) has enabled selective assays on complex mixtures to broaden the analyte scope. Droplet microfluidics has been interfaced to MS via electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The works reviewed herein outline the development of this nascent field as well as initial exploration of its application in biotechnology and bioanalysis, including synthetic biology, reaction development, and in vivo sensing.

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation.

Chemical modifications to protein encoding messenger RNAs (mRNAs) influence their localization, translation, and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. We have overcome these challenges by improving the mRNA purification and LC-MS/MS pipelines. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples, quantify 50 ribonucleosides in a single analysis, and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and reveal the presence of four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2, respectively), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells. To evaluate this possibility, we used a reconstituted translation system to investigate the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

Direct sequencing of total Saccharomyces cerevisiae tRNAs by LC-MS/MS.

Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant posttranscriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases, and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time, because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA-specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence Saccharomyces cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs.

Screening Clones for Monoclonal Antibody Production Using Droplet Microfluidics Interfaced to Electrospray Ionization Mass Spectrometry.

As one of the most critical steps in process development for protein therapeutics, clone selection and cell culture optimization require a large number of samples to be screened for high titer and desirable molecular profiles. Typical analytical techniques, such as chromatographic approaches, often take minutes per sample which are inefficient for large-scale screenings. Droplet microfluidics coupled to mass spectrometry (MS) represents an attractive approach due to its low volume requirements, high-throughput capabilities, label-free nature, and ability to handle complex mixtures. In this work, we coupled a modified protein cleanup protocol with a droplet-MS workflow for mAb titer screening to guide clone selection. With this droplet approach we achieved a throughput of 0.04 samples/s with an LoD of 0.15 mg/mL and an LoQ of 0.45 mg/mL. To test its performance in a real-world setting, this workflow was applied to a 35-clone screen, where the top 20% producing clones were identified. In addition, we coupled our sample cleanup protocol to a high-resolution MS and compared the glycan profiles of the high titer clones. This work demonstrates that droplet-MS provides a rapid way of clone screening and cell culture optimization based on titer and molecular structure of the expressed proteins. Future work is aimed at increasing the throughput and automation of this droplet-MS technique.