CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. IMPORTANCE Acinetobacter baumannii is found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.

Identification of a small-molecule RPL11 mimetic that inhibits tumor growth by targeting MDM2-p53 pathway.

BACKGROUND: Targeting ribosome biogenesis to activate p53 has recently emerged as a therapeutic strategy in human cancer. Among various ribosomal proteins, RPL11 centralizes the nucleolar stress-sensing pathway by binding MDM2, leading to MDM2 inactivation and p53 activation. Therefore, the identification of MDM2-binding RPL11-mimetics would be valuable for anti-cancer therapeutics. METHODS: Based on the crystal structure of the interface between RPL11 and MDM2, we have identified 15 potential allosteric modulators of MDM2 through the virtual screening. RESULTS: One of these compounds, named S9, directly binds MDM2 and competitively inhibits the interaction between RPL11 and MDM2, leading to p53 stabilization and activation. Moreover, S9 inhibits cancer cell proliferation in vitro and in vivo. Mechanistic study reveals that MDM2 is required for S9-induced G2 cell cycle arrest and apoptosis, whereas p53 contributes to S9-induced apoptosis. CONCLUSIONS: Putting together, S9 may serve as a lead compound for the development of an anticancer drug that specifically targets RPL11-MDM2-p53 pathway.

Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Assay for Quantitative Measurement of Therapeutic Antibody Cocktail REGEN-COV Concentrations in COVID-19 Patient Serum.

REGEN-COV is a cocktail of two human IgG1 monoclonal antibodies (REGN10933 + REGN10987) that targets severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and has shown great promise to reduce the SARS-CoV-2 viral load in COVID-19 patients enrolled in clinical studies. A liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS)-based method, combined with trypsin and rAspN dual enzymatic digestion, was developed for the determination of total REGN10933 and total REGN10987 concentrations in several hundreds of pharmacokinetic (PK) serum samples from COVID-19 patients participating in phase I, II, and III clinical studies. The performance characteristics of this bioanalytical assay were evaluated with respect to linearity, accuracy, precision, selectivity, specificity, and analyte stability before and after enzymatic digestion. The developed LC-MRM-MS assay has a dynamic range from 10 to 2000 µg/mL antibody drug in the human serum matrix, which was able to cover the serum drug concentration from day 0 to day 28 after drug administration in two-dose groups for the clinical PK study of REGEN-COV. The concentrations of REGEN-COV in the two-dose groups measured by the LC-MRM-MS assay were comparable to the concentrations measured by a fully validated electrochemiluminescence (ECL) immunoassay.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analysis of Citrus limon Response to Graft Inoculation by Candidatus Liberibacter asiaticus.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon (Citrus limon) plants were graft-inoculated with either CLas-infected or control (CLas-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with CLas infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between CLas-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of CLas-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analyses of Citrus sinensis (L.) Osbeck Graft-Inoculated with "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Prednisolone/prednisone as adrenocorticotropic hormone alternative for infantile spasms: a meta-analysis of randomized controlled trials.

AIM: To compare the efficacy and safety of prednisolone/prednisone and adrenocorticotropic hormone (ACTH) in the treatment of infantile spasms using a meta-analysis of randomized controlled trials (RCTs). METHOD: In a systematic literature search of electronic databases (MEDLINE, Embase, the Cochrane Library), we identified RCTs that assessed prednisolone/prednisone compared with ACTH/tetracosactide in patients with infantile spasms. The electroclinical response and adverse events were evaluated. RESULTS: Six RCTs (616 participants) were included in the meta-analysis. Compared with prednisolone/prednisone, ACTH/tetracosactide was not superior in terms of cessation of spasms at day 14 (relative risk 1.19, 95% confidence interval [CI] 0.74-1.92), day 42 (relative risk 1.02, 95% CI 0.63-1.65), and resolution of hypsarrhythmia on electroencephalogram (relative risk 1.14, 95% CI 0.71-1.81); the incidences of common adverse reactions caused by ACTH/tetracosactide were not lower than that of prednisolone/prednisone for irritability (relative risk 0.79, 95% CI 0.57-1.10), increased appetite (relative risk 0.78, 95% CI 0.57-1.08), weight gain (relative risk 0.86, 95% CI 0.56-1.32), and gastrointestinal upset (relative risk 0.60, 95% CI 0.35-1.02), though it seemed less frequent. INTERPRETATION: Prednisolone/prednisone elicits a similar electroclinical response as ACTH for infantile spasms, which indicates that it can be an alternative to ACTH for treating infantile spasms. What this paper adds Prednisolone/prednisone is as effective as adrenocorticotropic hormone (ACTH) in electroclinical response of infantile spasms. Prednisolone/prednisone and ACTH cause similar and tolerable adverse effects, whose incidences are comparable. High-dose prednisone/prednisolone might be preferable to low dose for achieving freedom from spasms.

Prednisolona/prednisona como alternativa a la hormona adrenocorticotrópica para los espasmos infantiles: un metanálisis de ensayos controlados aleatorios OBJETIVO: Comparar la eficacia y seguridad de la prednisolona/prednisona y la hormona adrenocorticotrópica (ACTH) en el tratamiento de los espasmos infantiles mediante un metanálisis de ensayos controlados aleatorios (ECA). MÉTODO: En una búsqueda sistemática en la literatura de bases de datos electrónicas (MEDLINE, Embase, Cochrane Library), identificamos ECA que evaluaban prednisolona/ prednisona en comparación con ACTH/tetracosactida en pacientes con espasmos infantiles. Se evaluaron la respuesta electro clínica y los eventos adversos. RESULTADOS: Seis ECA (616 participantes) se incluyeron en el metanálisis. En comparación con la prednisolona/prednisona, la ACTH/tetracosactida no fue superior en términos de cese de espasmos en el día 14 (riesgo relativo 1,19, intervalo de confianza del 95% [IC] 0,74-1,92), día 42 (riesgo relativo 1,02, IC del 95% 0,63- 1,65), y la resolución de la hipsarritmia en el EEG (riesgo relativo 1,14, IC 95% 0,71-1,81); la incidencia de reacciones adversas comunes causadas por ACTH/tetracosactida no fue inferior a la de prednisolona/prednisona para irritabilidad (riesgo relativo 0,79, IC 95% 0,57-11,10), aumento del apetito (riesgo relativo 0,78, IC 95% 0,57-1,08), aumento de peso (riesgo relativo 0,86; IC del 95%: 0,56-1,32) y malestar gastrointestinal (riesgo relativo 0,60; IC del 95%: 0,35-1,02), aunque parecía menos frecuente. INTERPRETACIÓN: La prednisolona/prednisona provoca una respuesta electro clínica similar a la ACTH para los espasmos infantiles, lo que indica que puede ser una alternativa a la ACTH para el tratamiento de los espasmos infantiles.

Prednisolona/predinisona como hormônio adrenocorticotrópico alternativo para espasmos infantis: uma metanálise de estudos randomizados controlados OBJETIVO: Comparar a eficácia e segurança da prednisolona/ prednisona e hormônio adrenocorticotrópio (HACT) no tratamento de espasmos infantis usando uma metanálise de estudos randomizados controlados (ERCs). MÉTODO: Em uma busca sistemática da literatura em bases de dados eletrônicas (MEDLINE, Embase, Biblioteca Cochrane), identificamos ERCs que avaliaram a prednisolona/ prednisona em comparação com o HACT/ tetracosactídeo em pacientes com espasmos infantis. A resposta eletroclínica e eventos adversos foram avaliados. RESULTADOS: Seis ERCs (616 participantes) foram incluídos na metanálise. Comparado com a prednisolona/ prednisona , o HACT/ tetracosactídeo não foi superior em termos de cessação dos espasmos no dia 14 (risco relativo 1,19, intervalo de confiança [IC] a 95% 0,74-1,92), dia 42 (risco relativo 1,02, IC 95% 0,63-1,65), e resolução da hipsarritimia no EEG (risco relativo 1,14, IC 95% 0,71-1,81); as incidências de reações adversas comuns causadas pelo HACT/ tetracosactídeo não foram menores que as da prednisolona/ prednisona para irritabilidade (risco relativo 0,79, IC 95% 0,57-1010), aumento do apetite (risco relativo 0,78, IC 95% 0,57-1,08), ganho de peso (risco relativo 0,86, IC 95% 0,56-1,32), e mal-estar gastrointestinal (risco relativo 0,60, IC 95% 0,35-1,02), embora parecessem menos frequentes. INTERPRETAÇÃO: A prednisolona/ prednisona /prednisone elicia resposta eletroclínica similar ao HACT para espasmos infantis, o que indica que pode ser uma alternativa ao HACD para tratar espasmos infantis.

Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags: A Probe for Quantitative Glycomic Analysis.

Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.

Targeted MultiNotch MS3 Approach for Relative Quantification of N-Glycans Using Multiplexed Carbonyl-Reactive Isobaric Tags.

The recently developed and commercially available carbonyl-reactive tandem mass tags (aminoxyTMT) enable multiplexed quantification of glycans through comparison of reporter ion intensities. However, challenges still exist for collision activated dissociation (CAD) MS/MS based quantification of aminoxyTMT due to the relatively low reporter ion yield especially for glycans with labile structures. To circumvent this limitation, we utilized the unique structural features of N-glycan molecules, the common core sugar sequence (HexNAc)2(Man)3, and common m/z of Yn ions generated from different types of precursors by MS/MS and designed a Y1 ion triggered, targeted MultiNotch MS3 relative quantification approach based on aminoxyTMT labeling. This approach was implemented on a nanoHILIC-Tribrid quadrupole-ion trap-Orbitrap platform, which enables prescreening of aminoxyTMT labeled N-glycan precursor ions by Y1 ion fragment ion mass in a higher-energy collisional dissociation (HCD) MS/MS scan and coisolation and cofragmentation of multiple Yn fragment ions that carry the isobaric tags from the inclusion list in the MS/MS/MS scan. Through systematical optimization and evaluation using N-glycans released from several glycoprotein standards and human serum proteins, we demonstrated that the Y1 ion triggered, targeted MultiNotch MS3 approach offers improved accuracy, precision, and sensitivity for relative quantification compared to traditional data-dependent MS2 and Y1 ion MS3 quantification methods.

Quantitative Glycomic Analysis by Mass-Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags and High-Resolution Mass Spectrometry.

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS1 level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS1 peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.

Profiling of small molecule metabolites and neurotransmitters in crustacean hemolymph and neuronal tissues using reversed-phase LC-MS/MS.

Crustaceans have been long used as model animals for neuromodulation studies because of their well-defined neural circuitry. The identification of small molecule metabolites and signaling molecules in circulating fluids and neuronal tissues presents unique challenges due to their diverse structures, biological functions, and wide range of concentrations. LC combined with high resolution MS/MS is one of the most powerful tools to uncover endogenous small molecules. Here we explored several sample preparation techniques (solid-phase extraction and denaturing) and MS data acquisition strategies (data-dependent acquisition and targeted MS2-based acquisition) that provided complementary coverage and improved overall identification rate in C18 LC-MS/MS experiment. By MS/MS spectral matching with mzCloud database and those generated from standard compounds, a total of 129 small molecule metabolites and neurotransmitters were identified from crustacean hemolymph and neuronal tissues. These confidently identified small molecules covered predominant biosynthetic pathways for major neurotransmitters, validating the effectiveness of the high-throughput RPLC-MS/MS approach in studying the metabolism of neurotransmitters.

A Novel Feature Optimization for Wearable Human-Computer Interfaces Using Surface Electromyography Sensors.

The novel human-computer interface (HCI) using bioelectrical signals as input is a valuable tool to improve the lives of people with disabilities. In this paper, surface electromyography (sEMG) signals induced by four classes of wrist movements were acquired from four sites on the lower arm with our designed system. Forty-two features were extracted from the time, frequency and time-frequency domains. Optimal channels were determined from single-channel classification performance rank. The optimal-feature selection was according to a modified entropy criteria (EC) and Fisher discrimination (FD) criteria. The feature selection results were evaluated by four different classifiers, and compared with other conventional feature subsets. In online tests, the wearable system acquired real-time sEMG signals. The selected features and trained classifier model were used to control a telecar through four different paradigms in a designed environment with simple obstacles. Performance was evaluated based on travel time (TT) and recognition rate (RR). The results of hardware evaluation verified the feasibility of our acquisition systems, and ensured signal quality. Single-channel analysis results indicated that the channel located on the extensor carpi ulnaris (ECU) performed best with mean classification accuracy of 97.45% for all movement's pairs. Channels placed on ECU and the extensor carpi radialis (ECR) were selected according to the accuracy rank. Experimental results showed that the proposed FD method was better than other feature selection methods and single-type features. The combination of FD and random forest (RF) performed best in offline analysis, with 96.77% multi-class RR. Online results illustrated that the state-machine paradigm with a 125 ms window had the highest maneuverability and was closest to real-life control. Subjects could accomplish online sessions by three sEMG-based paradigms, with average times of 46.02, 49.06 and 48.08 s, respectively. These experiments validate the feasibility of proposed real-time wearable HCI system and algorithms, providing a potential assistive device interface for persons with disabilities.

Large-Scale and Targeted Quantitative Cross-Linking MS Using Isotope-Labeled Protein Interaction Reporter (PIR) Cross-Linkers.

Quantitative measurement of chemically cross-linked proteins is crucial to reveal dynamic information about protein structures and protein-protein interactions and how these are differentially regulated during stress, aging, drug treatment, and most perturbations. Previously, we demonstrated how quantitative in vivo cross-linking (CL) with stable isotope labeling of amino acids in cell culture (SILAC) enables both heritable and dynamic changes in cells to be visualized. In this work, we demonstrate the technical feasibility of proteome-scale quantitative in vivo CL-MS using isotope-labeled protein interaction reporter (PIR) cross-linkers and E. coli as a model system. This isotope-labeled cross-linkers approach, combined with Real-time Analysis of Cross-linked peptide Technology (ReACT) previously developed in our lab, enables the quantification of 941 nonredundant cross-linked peptide pairs from a total of 1213 fully identified peptide pairs in two biological replicate samples through comparison of MS1 peak intensity of the light and heavy cross-linked peptide pairs. For targeted relative quantification of cross-linked peptide pairs, we further developed a PRM-based assay to accurately probe specific site interaction changes in a complex background. The methodology described in this work provides reliable tools for both large-scale and targeted quantitative CL-MS that is useful for any sample where SILAC labeling may not be practical.

Development of a hydrophilic interaction liquid chromatography coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging platform for N-glycan relative quantitation using stable-isotope labeled hydrazide reagents.

In this work, the capability of newly developed hydrophilic interaction liquid chromatography (HILIC) coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) platform for quantitative analysis of N-glycans has been demonstrated. As a proof-of-principle experiment, heavy and light stable-isotope labeled hydrazide reagents labeled maltodextrin ladder were used to demonstrate the feasibility of the HILIC-MALDI-MSI platform for reliable quantitative analysis of N-glycans. MALDI-MSI analysis by an Orbitrap mass spectrometer enabled high-resolution and high-sensitivity detection of N-glycans eluted from HILIC column, allowing the re-construction of LC chromatograms as well as accurate mass measurements for structural inference. MALDI-MSI analysis of the collected LC traces showed that the chromatographic resolution was preserved. The N-glycans released from human serum was used to demonstrate the utility of this novel platform in quantitative analysis of N-glycans from a complex sample. Benefiting from the minimized ion suppression provided by HILIC separation, comparison between MALDI-MS and the newly developed platform HILIC-MALDI-MSI revealed that HILIC-MALDI-MSI provided higher N-glycan coverage as well as better quantitation accuracy in the quantitative analysis of N-glycans released from human serum. Graphical abstract Reconstructed chromatograms based on HILIC-MALDI-MSI results of heavy and light labeled maltodextrin enabling quantitative glycan analysis.

Quantitative analysis of serotonin secreted by human embryonic stem cells-derived serotonergic neurons via pH-mediated online stacking-CE-ESI-MRM.

A CE-ESI-MRM-based assay was developed for targeted analysis of serotonin released by human embryonic stem cells-derived serotonergic neurons in a chemically defined environment. A discontinuous electrolyte system was optimized for pH-mediated online stacking of serotonin. Combining with a liquid-liquid extraction procedure, LOD of serotonin in the Krebs'-Ringer's solution by CE-ESI-MS/MS on a 3D ion trap MS was0.15 ng/mL. The quantitative results confirmed the serotonergic identity of the in vitro developed neurons and the capacity of these neurons to release serotonin in response to stimulus.

Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry for Quantitative Analysis of Glycans Labeled with Multiplex Carbonyl-Reactive Tandem Mass Tags.

Recently developed carbonyl-reactive aminoxy tandem mass tag (aminoxyTMT) reagents enable multiplexed characterization and quantitative comparison of structurally complex glycans between different biological samples. Compared to some previously reported isotopic labeling strategies for glycans, the use of the aminoxyTMT method features a simple labeling procedure, excellent labeling efficiency, and reduced spectral complexity at the MS(1) level. Presence of the tertiary amine functionality in the reporter region of the aminoxyTMT labels leads to increased ionization efficiency of the labeled glycans thus improving electrospray ionization (ESI)-mass spectrometry (MS) detection sensitivity. The use of the labeling reagent also makes electrophoretic separation of the labeled neutral and acidic glycans feasible. In this work, we characterized the ESI and collision induced dissociation (CID) behavior of the aminoxyTMT-labeled neutral and sialylated glycans. For the high-mannose N-glycans and small sialylated oligosaccharides, CID fragmentation of [M + Na + H](2+) provides the most informative MS(2) spectra for both quantitative and qualitative analysis. For complex N-glycans, MS(3) of the protonated Y1(H) ion can be used for relative quantification without interference from the HexNAc fragments. Online capillary electrophoresis (CE)-ESI-MS/MS analyses of multiplexed aminoxyTMT-labeled human milk oligosaccharides (HMOs) and different types of N-glycans released from glycoprotein standards were demonstrated. Improved resolution and quantification accuracy of the labeled HMO isomers was achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. N-Glycans released from human serum protein digests were labeled with six-plex aminoxyTMT and subjected to CE-ESI-MS/pseudo-MS(3) analysis, which demonstrated the potential utility of this glycan relative quantification platform for more complex biological samples.

Relative quantification of amine-containing metabolites using isobaric N,N-dimethyl leucine (DiLeu) reagents via LC-ESI-MS/MS and CE-ESI-MS/MS.

Tandem mass spectrometry (MS/MS)-based relative quantification by isobaric labeling is a useful technique to compare different metabolic expression levels in biological systems. For the first time, we have labeled primary and secondary amine-containing small molecules using 4-plex isobaric N,N-dimethyl leucine (DiLeu) to perform relative quantification. Good labeling efficiency and quantification accuracy were demonstrated with a mixture of 12 metabolite standards including amino acids and small molecule neurotransmitters. Labeling amine-containing metabolites with DiLeu reagents also enabled the separation of polar metabolites by nanoRPLC and improved the detection sensitivity by CE-ESI-MS. The 4-plex DiLeu labeling technique combined with LC-MS/MS and CE-MS/MS platforms were applied to profile and quantify amine-containing metabolites in mouse urine. The variability of concentrations of identified metabolites in urine samples from different mouse individuals was illustrated by the ratios of reporter ion intensities acquired from online data-dependent analysis.

Reversible posterior encephalopathy syndrome in children with nephrotic syndrome.

AIM: To investigate the clinical features and prognoses of children who develop reversible posterior encephalopathy syndrome (RPES) during treatment for nephrotic syndrome (NS). METHODS: The clinicoradiological characteristics and prognoses of 51 patients with NS, including 21 with RPES and 30 without, were analyzed. RESULTS: Compared with the controls, the RPES patients exhibited a higher rate of tacrolimus (P = 0.01) and cyclosporine (P = 0.02) treatment; higher-dose prednisolone (P = 0.01) treatment; higher systolic blood pressure (P = 0.04), serum cholesterol (P = 0.03), and proteinuria (P < 0.01); and lower serum albumin levels (P = 0.03). Hypertension was present in 85.7% of RPES patients. The clinical manifestations of RPES included an altered mental status, seizures, headaches, nausea and vomiting, and visual impairment. Electroencephalography findings included slow waves and focal sharp or/and spiked waves; magnetic resonance imaging showed lesions localized in the occipital, parietal, frontal, temporal lobes and the cerebellum and brainstem; and magnetic resonance angiography revealed vertebral artery narrowing. All RPES patients recovered completely with timely and appropriate therapy. CONCLUSION: Hypertension, calcineurin inhibitor and high-dose steroid treatments, high serum cholesterol and proteinuria levels, and low serum albumin levels can predispose children with NS to RPES, although both the clinical and imaging outcomes are satisfactory.

Recent advances in enrichment and separation strategies for mass spectrometry-based phosphoproteomics.

Due to the significance of protein phosphorylation in various biological processes and signaling events, new analytical techniques for enhanced phosphoproteomics have been rapidly introduced in the recent years. The combinatorial use of the phospho-specific enrichment techniques and prefractionation methods prior to MS analysis enable comprehensive profiling of the phosphoproteome and facilitate deciphering the critical roles that phosphorylation plays in signaling pathways in various biological systems. This review places special emphasis on the recent five-year (2009-2013) advances for enrichment and separation techniques that have been utilized for phosphopeptides prior to MS analysis.

Recent advances in coupling capillary electrophoresis-based separation techniques to ESI and MALDI-MS.

Coupling CE-based separation techniques to MS creates a powerful platform for analysis of a wide range of biomolecules from complex samples because it combines the high separation efficiency of CE and the sensitivity and selectivity of MS detection. ESI and MALDI, as the most common soft ionization techniques employed for CE and MS coupling, offer distinct advantages for biomolecular characterization. This review is focused primarily on technological advances in combining CE and chip-based CE with ESI and MALDI-MS detection in the past five years. Selected applications in the analyses of metabolites, peptides, and proteins with recently developed CE-MS platforms are also highlighted.

Kinetics of Trisulfide-to-Disulfide Conversion of Therapeutic IgG1 Monoclonal Antibodies Under Physiological Conditions: A Case Study of Casirivimab And Imdevimab.

The percentage of trisulfide variants is a product quality metric that is monitored during the manufacture of monoclonal antibody (mAb)-based therapeutics. Results from earlier preclinical studies revealed that trisulfide linkages in mAbs are rapidly converted to disulfides in circulation. In this study, casirivimab and imdevimab, which are both IgG1 subclass mAbs that target the non-overlapping epitopes in SARS-CoV2 Spike protein, are used as models to study the kinetics of trisulfide-to-disulfide conversion in vivo in human circulation. To determine the percentage of trisulfide variants in systemic circulation immediately after intravenous injection, both mAbs were immunoprecipitated from serum samples collected from COVID-19 patients that received this cocktail antibody treatment as part of a first-in-human study. The immunoprecipitated mAbs were then digested under non-reducing conditions and evaluated by liquid-chromatography-mass spectrometry (LC-MS). Significant reductions in the percentages of trisulfide variants were observed in serum samples as early as 1 hr after completion of the intravenous infusion. A flow-through dialysis model designed to mimic the redox potential of blood revealed a plausible chemical mechanism for the rapid trisulfide-to-disulfide conversion of IgG1 subclass mAbs under physiological conditions.