High-throughput Mass Spectrometry Research Articles

Omics-Based Investigations of Breast Cancer.

Breast cancer (BC) is characterized by an extensive genotypic and phenotypic heterogeneity. In-depth investigations into the molecular bases of BC phenotypes, carcinogenesis, progression, and metastasis are necessary for accurate diagnoses, prognoses, and therapy assessments in predictive, precision, and personalized oncology. This review discusses both classic as well as several novel omics fields that are involved or should be used in modern BC investigations, which may be integrated as a holistic term, onco-breastomics. Rapid and recent advances in molecular profiling strategies and analytical techniques based on high-throughput sequencing and mass spectrometry (MS) development have generated large-scale multi-omics datasets, mainly emerging from the three "big omics", based on the central dogma of molecular biology: genomics, transcriptomics, and proteomics. Metabolomics-based approaches also reflect the dynamic response of BC cells to genetic modifications. Interactomics promotes a holistic view in BC research by constructing and characterizing protein-protein interaction (PPI) networks that provide a novel hypothesis for the pathophysiological processes involved in BC progression and subtyping. The emergence of new omics- and epiomics-based multidimensional approaches provide opportunities to gain insights into BC heterogeneity and its underlying mechanisms. The three main epiomics fields (epigenomics, epitranscriptomics, and epiproteomics) are focused on the epigenetic DNA changes, RNAs modifications, and posttranslational modifications (PTMs) affecting protein functions for an in-depth understanding of cancer cell proliferation, migration, and invasion. Novel omics fields, such as epichaperomics or epimetabolomics, could investigate the modifications in the interactome induced by stressors and provide PPI changes, as well as in metabolites, as drivers of BC-causing phenotypes. Over the last years, several proteomics-derived omics, such as matrisomics, exosomics, secretomics, kinomics, phosphoproteomics, or immunomics, provided valuable data for a deep understanding of dysregulated pathways in BC cells and their tumor microenvironment (TME) or tumor immune microenvironment (TIMW). Most of these omics datasets are still assessed individually using distinct approches and do not generate the desired and expected global-integrative knowledge with applications in clinical diagnostics. However, several hyphenated omics approaches, such as proteo-genomics, proteo-transcriptomics, and phosphoproteomics-exosomics are useful for the identification of putative BC biomarkers and therapeutic targets. To develop non-invasive diagnostic tests and to discover new biomarkers for BC, classic and novel omics-based strategies allow for significant advances in blood/plasma-based omics. Salivaomics, urinomics, and milkomics appear as integrative omics that may develop a high potential for early and non-invasive diagnoses in BC. Thus, the analysis of the tumor circulome is considered a novel frontier in liquid biopsy. Omics-based investigations have applications in BC modeling, as well as accurate BC classification and subtype characterization. The future in omics-based investigations of BC may be also focused on multi-omics single-cell analyses.

High-Throughput Analysis of Underivatized Amino Acids and Acylcarnitines in Infant Serum: A Micromethod Based on Stable Isotope Dilution Targeted HILIC-ESI-MS/MS.

Amino acids and acylcarnitines are important biomarkers of the body's energy state and can be used as diagnostic markers of certain inborn errors of metabolism. Few multianalyte methods for high-throughput analysis in serum exist for these compounds, but micromethods suitable for use in young children and infants are lacking. Therefore, we developed a quantitative high-throughput multianalyte hydrophilic interaction liquid chromatography-tandem mass spectrometry method preceded by a derivatization-free sample preparation using minimum amounts of serum (25 μL). Isotopically labeled standards were utilized for quantification. Forty amino acids and amino acid derivatives and 22 acylcarnitines were detected by applying a multiple reaction monitoring mode within a 20 min run. The method was comprehensively validated, comprising linearity, accuracy, (intraday/interday) precision, and quantitation limits, of which the latter ranged from 0.25 to 50 nM for acylcarnitines and from 0.005 to 1 μM for amino acids and their derivatives. Application of the method to 145 serum samples of three- to four-month-old healthy infants showed excellent reproducibility for multiday analyses and enabled simultaneous amino acid and acylcarnitine profiling in this age group.

Profile and potential role of novel metabolite biomarkers, especially indoleacrylic acid, in pathogenesis of neuromyelitis optica spectrum disorders.

Background: Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune central nervous system (CNS) inflammatory and demyelinating disorder that can lead to serious disability and mortality. Humoral fluid biomarkers with specific, convenient, and efficient profiles that could characterize and monitor disease activity or severity are very useful. We aimed to develop a sensitive and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS)/MS-based analytical method for novel biomarkers finding in NMOSD patients and verified its function tentatively. Methods: Serum samples were collected from 47 NMOSD patients, 18 patients with other neurological disorders (ONDs), and 35 healthy controls (HC). Cerebrospinal fluid (CSF) samples were collected from 18 NMOSD and 17 OND patients. Three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) and nine important metabolites that included phenylacetylglutamine (PAGln), indoleacrylic acid (IA), 3-indole acetic acid (IAA), 5-hydroxyindoleacetic acid (HIAA), hippuric acid (HA), I-3-carboxylic acid (I-3-CA), kynurenine (KYN), kynurenic acid (KYNA), and quinine (QUIN) were analyzed by using the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. The profile of IA was further analyzed, and its function was verified in an astrocyte injury model stimulated by NMO-IgG, which represents important events in NMOSD pathogenesis. Results: In the serum, tyrosine and some of the tryptophan metabolites IA and I-3-CA decreased, and HIAA increased significantly in NMOSD patients. The CSF levels of phenylalanine and tyrosine showed a significant increase exactly during the relapse stage, and IA in the CSF was also increased markedly during the relapse and remission phases. All conversion ratios had similar profiles with their level fluctuations. In addition, the serum IA levels negatively correlated with glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) levels in the serum of NMOSD patients were measured by using ultra-sensitive single-molecule arrays (Simoa). IA showed an anti-inflammatory effect in an in vitro astrocyte injury model. Conclusion: Our data suggest that essential aromatic amino acid tryptophan metabolites IA in the serum or CSF may serve as a novel promising biomarker to monitor and predict the activity and severity of NMOSD disease. Supplying or enhancing IA function can promote anti-inflammatory responses and may have therapeutic benefits.

Applying Automation and High-Throughput MALDI Mass Spectrometry for Peptide Metabolic Stability Screening

The discovery of peptide therapeutics represents a fast-growing segment of pharmaceutical research. During the early discovery process, a large number of peptide candidates needs to be rapidly screened for metabolic stability in relevant biological matrices. In most cases, peptide stability assays are quantified using LC-MS/MS, which may take hours to analyze 384 samples and generates liters of solvent waste. Herein, we introduce a high-throughput screening (HTS) platform for peptide stability assessment founded on Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS). Full automation has been implemented for sample preparation with minimal manual intervention. The limit of detection, linearity, and reproducibility of the platform were evaluated, and metabolic stabilities have been determined for a number of peptide candidates. The MALDI-MS-based HTS workflow is able to analyze 384 samples in less than 1 h while only using 115 μL of total solvent. Although this process allows for very rapid assessment of peptide stability, given the nature of the MALDI process, it is noteworthy that spot-to-spot variations and ionization bias are observed. Therefore, LC-MS/MS may still be needed for confident, quantitative measurements and/or when the ionization efficiency of certain peptides is inadequate using MALDI.

Automated high-throughput characterization of microbial metabolites and species using array ballpoint electrospray ionization technique.

Microbial metabolites are widely used in agriculture, food, environment, and medicine. However, there is a lack of high-throughput, nonclogging, and simple methods for the identification of microbial metabolites and their subspecies using ambient mass spectrometry (MS). Here, we proposed a method for analyzing the microbial metabolites and identifying their species using the array ballpoint electrospray ionization (aBPESI) technique. The previously developed BPESI was combined with the array analysis technique to form a high-throughput analysis technique called aBPESI. The bacteria cultured on the plate medium were directly analyzed using MS with aBPESI. Principal component analysis-linear discriminant analysis (PCA-LDA)algorithm was used to analyze the different subspecies groups. The results showed that aBPESI was capable of completing a sample analysis within 30 s, and the level of metabolite detection was comparable to existing techniques. The accuracy of bacterial subspecies identification was 90% (Pseudomonas aeruginosa) and 100% (Serratia marcescens). A new high-throughput and robust MS technique aBPESI was proposed. It does not require sample pretreatment and greatly reduces the sample analysis time. aBPESI shows a strong ability in microbial analysis and is expected to be further applied in other research fields.

Proteomic profiling of retina and retinal pigment epithelium combined embryonic tissue to facilitate ocular disease gene discovery.

To expedite gene discovery in eye development and its associated defects, we previously developed a bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery). However, iSyTE is presently limited to lens tissue and is predominantly based on transcriptomics datasets. Therefore, to extend iSyTE to other eye tissues on the proteome level, we performed high-throughput tandem mass spectrometry (MS/MS) on mouse embryonic day (E)14.5 retina and retinal pigment epithelium combined tissue and identified an average of 3300 proteins per sample (n = 5). High-throughput expression profiling-based gene discovery approaches-involving either transcriptomics or proteomics-pose a key challenge of prioritizing candidates from thousands of RNA/proteins expressed. To address this, we used MS/MS proteome data from mouse whole embryonic body (WB) as a reference dataset and performed comparative analysis-termed "in silico WB-subtraction"-with the retina proteome dataset. In silico WB-subtraction identified 90 high-priority proteins with retina-enriched expression at stringency criteria of ≥ 2.5 average spectral counts, ≥ 2.0 fold-enrichment, false discovery rate < 0.01. These top candidates represent a pool of retina-enriched proteins, several of which are associated with retinal biology and/or defects (e.g., Aldh1a1, Ank2, Ank3, Dcn, Dync2h1, Egfr, Ephb2, Fbln5, Fbn2, Hras, Igf2bp1, Msi1, Rbp1, Rlbp1, Tenm3, Yap1, etc.), indicating the effectiveness of this approach. Importantly, in silico WB-subtraction also identified several new high-priority candidates with potential regulatory function in retina development. Finally, proteins exhibiting expression or enriched-expression in the retina are made accessible in a user-friendly manner at iSyTE ( https://research.bioinformatics.udel.edu/iSyTE/ ), to allow effective visualization of this information and facilitate eye gene discovery.

“Lab of the Future”─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics

Here we describe a state-of-the-art, integrated, multi-instrument automated system designed to execute methods involved in mass spectrometry characterization of biotherapeutics. The system includes liquid and microplate handling robotics and utilities, integrated LC-MS, along with data analysis software, to perform sample purification, preparation, and analysis as a seamless integrated unit. The automated process begins with tip-based purification of target proteins from expression cell-line supernatants, which is initiated once the samples are loaded onto the automated system and the metadata are retrieved from our corporate data aggregation system. Subsequently, the purified protein samples are prepared for MS, including deglycosylation and reduction steps for intact and reduced mass analysis, and proteolytic digestions, desalting, and buffer exchange via centrifugation for peptide map analysis. The prepared samples are then loaded into the LC-MS instrumentation for data acquisition. The acquired raw data are initially stored on a local area network storage system that is monitored by watcher scripts that then upload the raw MS data to a network of cloud-based servers. The raw MS data are processed with the appropriately configured analysis workflows such as database search for peptide mapping or charge deconvolution for undigested proteins. The results are verified and formatted for expert curation directly in the cloud. Finally, the curated results are appended to sample metadata in the corporate data aggregation system to accompany the biotherapeutic cell lines in subsequent processes.

Metabolomics in plant pathogen-defence: from single molecules to large scale analysis.

Plants produce a high diversity of secondary metabolites that are involved in a wide range of different functions, including stress tolerance, signaling molecule for interactions with other species (allelopathy) and protecting plants against herbivores and pathogens. With the rise of more accessible, high-throughput mass spectrometry and new analytical tools it becomes feasible to identify and validate new secondary metabolites involved in pathogen resistance or assign new roles to previously detected compounds. In this review, we provide a brief overview of the major pathogen defence-associated classes of secondary metabolites, with a focus on those with direct anti-pathogen function. For each class we highlight one or more typical examples representing the class to give a comprehensive summary of some of the work done to date. In the second part of this review, we highlight how new technological advances and high throughput experiments in combination with other sources of -omics data, like genomics and transcriptomics can accelerate the studies on secondary metabolites and help to link these compounds to genotypes. Employing such approaches will improve our understanding of chemical defences against plant pathogens and allow rapid development of markers for resistance breeding.

Square Membrane Resonators Supporting Degenerate Modes of Vibration for High-Throughput Mass Spectrometry of Single Bacterial Cells.

In nanomechanical mass spectrometry, sensing devices are commonly placed in the vacuum environment and a stream of analytes is directed toward the sensor surface for measurement. Beam structures, such as double-clamped nanobeams and nanocantilevers, are commonly used due to their low inertial mass and the simplicity of the analytical models for mass extraction. The drawback of such structures is their low capture areas, compromising the capture efficiency and throughput of this technique. Bi-axisymmetric resonators, such as ultrathin square or circular membranes, arise as an optimal geometry to maximize capture efficiency while minimizing the device inertial mass. However, these structures present degenerate mechanical modes, whose frequency perturbations upon analyte adsorption are not well described by commonly used models. Furthermore, prior knowledge of the vibration mode shapes of the sensor is crucial for the correct calculation of the analyte's mass, and the mode shape of degenerate modes may change significantly after every adsorption event. In this work, we present an accurate analytical theory to describe the effect of mass adsorption on the degenerate modes of square membrane resonators and propose two different methods based on the new theory to update the vibration mode shapes after every adsorption event. Finally, we illustrate the problem experimentally obtaining the mass and adsorption position of individual Escherichia coli K-12 bacterial cells on commercial square silicon nitride membranes fabricated with very small tolerances.

Pharmaceutical Analysis in Drug Discovery and Drug Development

Pharmaceutical analysis stands as a linchpin within the intricate and multifaceted landscape of drug development. It assumes a pivotal role in upholding the safety, efficacy and quality of medicinal products at each juncture of their odyssey from conceptualization to market integration. Within this discourse, we delve into the paramount role of pharmaceutical analysis within the sphere of drug development. Pharmaceutical analysis plays an instrumental role in the identification and characterization of lead compounds that harbor therapeutic potential. Techniques such as high-throughput screening, mass spectrometry and nuclear magnetic resonance (NMR) serve as guiding lights in the quest for promising candidates. The elucidation of molecular structures through the means of NMR and X-ray crystallography takes center stage, facilitating a profound comprehension of the intricate interplay between drugs and biological targets. In this review we focused on role of pharmaceutical analysis in drug development and drug discovery. Inadequate pharmaceutical analysis and manufacturing practices can lead to the distribution of unsafe and ineffective medicines. Thus, adherence to cGMP is not only a legal obligation but also a moral imperative to protect public health and maintain the integrity of the pharmaceutical industry.

Ultra-High-Throughput and Low-Volume Analysis of Intact Proteins with LAP-MALDI MS.

High-throughput (HTP) mass spectrometry (MS) is a rapidly growing field, with many techniques evolving to accommodate ever increasing sample analysis rates. Many of these techniques, such as AEMS and IR-MALDESI MS, require volumes of at least 20-50 μL for analysis. Here, liquid atmospheric pressure-matrix-assisted laser desorption/ionization (LAP-MALDI) MS is presented as an alternative for ultra-high-throughput analysis of proteins requiring only femtomole quantities of protein in 0.5 μL droplets. By moving a 384-well microtiter sample plate with a high-speed XY-stage actuator, sample acquisition rates of up to 10 samples per second have been achieved at a data acquisition rate of 200 spectra per scan. It is shown that protein mixture solutions with concentrations of ≤2 μM can be analyzed at this speed, while individual protein solutions can be analyzed at concentrations of ≤0.2 μM. Thus, LAP-MALDI MS provides a promising platform for multiplexed HTP protein analysis.

High-Throughput Nanoliter Sampling and Direct Analysis of Biological Fluids Using Droplet Imbibition Mass Spectrometry.

A high-throughput droplet imbibition mass spectrometry (MS) experiment is reported for the first time that allows direct analysis of ultra-small volumes of complex mixtures. In this experiment, an array of optimized tips of glass capillaries containing the analyte solution is sampled by rapidly moving charged microdroplets, which picks up (i.e., imbibes) the analyte and transfers it to a proximal mass spectrometer. The advantages associated with this droplet imbibition experiment include (1) ultra-small sample consumption (1.3 nL/min), which reduces the matrix effect in complex mixture analysis, and (2) high surface activity, which eliminates ion suppression effects caused by competition for the space charge on the droplet surface. Collectively, the enhanced surface effect and small flow rates dramatically increase the sensitivity of the droplet imbibition MS approach. This was experimentally shown by constructing calibration curves for cocaine analysis in human raw urine and whole blood, achieving 2 and 7 pg/mL limits of detection, respectively. The high-throughput feature was demonstrated by analyzing five structurally different compounds in 20 s intervals. With the measured flow rate of 1.3 nL/min on a 5 μm glass tip size, the results described in the current study showcase droplet imbibition MS to be a powerful and high-throughput alternative for conventional nano-electrospray ionization (flow rate <100 nL/min), which is the most efficient method for transferring small sample volumes to mass spectrometers.

Isobaric Stable Isotope N-Phosphorylation Labeling (iSIPL) for Ultrasensitive Proteome Quantification.

Stable isotope chemical labeling methods have been widely used for high-throughput mass spectrometry (MS)-based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P-N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV-1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat-induced activation of HIV-1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein-protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large-scale accurate quantitative proteomics by increasing multiplexing capability.

A high-throughput LC-MS/MS method for simultaneous determination of direct oral anticoagulants in human plasma.

Direct oral anticoagulants are widely used in many indications to prevent thromboembolic events. Routine therapeutic monitoring is not required; however, there is increasing evidence suggesting the benefit of plasma level measurement in some situations. In addition, laboratory monitoring might help improve patient and drug non-compliance and thus individualize therapy. In the present study, we developed a sensitive and high throughput ultra-high-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma. A one-step extraction procedure in 96-well formate for phospholipid and protein removal was used for sample pre-treatment, and analytes were separated using gradient elution over 4.2 min. Analytes were detected on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode. The method was validated according to the European Medicine Agency guideline for the selectivity, linearity and lower limit of detection, precision and accuracy, matrix effects, extraction recovery, carryover, dilution integrity and stability over a concentration range of 3.0-1000 ng/mL for all analytes. The validated method was applied to real clinical samples of patients treated with one of the drugs. Therefore, we can conclude that our method is suitable for therapeutic drug monitoring of direct oral anticoagulants. This article is protected by copyright. All rights reserved.

Staphylococcus aureus Cell Wall Phenotypic Changes Associated with Biofilm Maturation and Water Availability: A Key Contributing Factor for Chlorine Resistance

Staphylococcus aureus biofilms are resistant to both antibiotics and disinfectants. As Staphylococci cell walls are an important defence mechanism, we sought to examine changes to the bacterial cell wall under different growth conditions. Cell walls of S. aureus grown as 3-day hydrated biofilm, 12-day hydrated biofilm, and 12-day dry surface biofilm (DSB) were compared to cell walls of planktonic organisms. Additionally, proteomic analysis using high-throughput tandem mass tag-based mass spectrometry was performed. Proteins involved in cell wall synthesis in biofilms were upregulated in comparison to planktonic growth. Bacterial cell wall width (measured by transmission electron microscopy) and peptidoglycan production (detected using a silkworm larva plasma system) increased with biofilm culture duration (p < 0.001) and dehydration (p = 0.002). Similarly, disinfectant tolerance was greatest in DSB, followed by 12-day hydrated biofilm and then 3-day biofilm, and it was least in the planktonic bacteria--suggesting that changes to the cell wall may be a key factor for S. aureus biofilm biocide resistance. Our findings shed light on possible new targets to combat biofilm-related infections and hospital dry surface biofilms.

A rapid and sensitive single-cell proteomic method based on fast liquid-chromatography separation, retention time prediction and MS1-only acquisition

Single-cell analysis has received much attention in recent years for elucidating the widely existing cellular heterogeneity in biological systems. However, the ability to measure the proteome in single cells is still far behind that of transcriptomics due to the lack of sensitive and high-throughput mass spectrometry methods. Herein, we report an integrated strategy termed “SCP-MS1” that combines fast liquid chromatography (LC) separation, deep learning-based retention time (RT) prediction and MS1-only acquisition for rapid and sensitive single-cell proteome analysis. In SCP-MS1, the peptides were identified via four-dimensional MS1 feature (m/z, RT, charge and FAIMS CV) matching, therefore relieving MS acquisition from the time consuming and information losing MS2 step and making this method particularly compatible with fast LC separation. By completely omitting the MS2 step, all the MS analysis time was utilized for MS1 acquisition in SCP-MS1 and therefore led to 65%–138% increased MS1 feature collection. Unlike “match between run” methods that still needed MS2 information for RT alignment, SCP-MS1 used deep learning-based RT prediction to transfer the measured RTs in long gradient bulk analyses to short gradient single cell analyses, which was the key step to enhance both identification scale and matching accuracy. Using this strategy, more than 2000 proteins were obtained from 0.2 ng of peptides with a 14-min active gradient at a false discovery rate (FDR) of 0.8%. Comparing with the DDA method, improved quantitative performance was also observed for SCP-MS1 with approximately 50% decreased median coefficient of variation of quantified proteins. For single-cell analysis, 1715 ± 204 and 1604 ± 224 proteins were quantified in single 293T and HeLa cells, respectively. Finally, SCP-MS1 was applied to single-cell proteome analysis of sorafenib resistant and non-resistant HepG2 cells and revealed clear cellular heterogeneity in the resistant population that may be masked in bulk studies.

Steroid profiling for the diagnosis of congenital adrenal hyperplasia by microbore ultra-performance liquid chromatography–tandem mass spectrometry

BackgroundA rapid and accurate measurement approach for 17α-hydroxyprogesterone (17-OHP) and related steroids in amount/volume-limited clinic samples is of importance for precise newborn diagnosis of congenital adrenal hyperplasia (CAH) and its subtypes in clinic. MethodsSixteen steroids (17-OHP, androstenedione, cortisol, tetrahydro-11-deoxycortisol, pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 21-deoxycortisol, 11-deoxycortisol, dehydroepiandrosterone, testosterone, aldosterone, 17α-hydroxypregnenolone, dihydrotestosterone and 18-hydroxycorticosterone) were included in the panel of high-throughput microbore ultra-performance liquid chromatography-tandem mass spectrometry. Samples were collected from 126 normal subjects and 65 patients including different subtypes of CAH. ResultsThe method was validated with satisfactory analytical performance in linearity, repeatability, recovery and limit of detection. Reference intervals for 16 steroids were established by quantifying the level of steroids detected in normal infants. The applicability of the method was tested by differentiating steroid metabolic characteristics between normal infants and infants with CAH, as well as between infants with different CAH subtypes. The relevance of 17-OHP, 21-deoxycortisol, and 17-OHP/11-deoxycortisol for 21-hydroxylase deficiency screening was demonstrated. The level of 11-deoxycorticosterone, 11-deoxycortisol, progesterone and androstenedione can be used for the diagnosis of different rare subtypes of CAH. ConclusionThis study provides a strategy for highly efficient steroid analysis of amount/volume-limited clinic samples and holds great potential for clinical diagnosis of CAH.

Comprehensive Comparisons between Grafted Kynam Agarwood and Normal Agarwood on Traits, Composition, and In Vitro Activation of AMPK.

Agarwood, a highly valuable resin/wood combination with diverse pharmacological activities but scarce supply, has a long history of being used as a medicine in several medical systems. Grafted Kynam agarwood (GKA) has been cultivated successfully recently and has the qualities meeting the definition of premium Kynam agarwood. However, there are few comprehensive comparisons between GKA and normal agarwood in terms of traits, global composition, and activity, and some key issues for GKA to be adopted into the traditional Chinese medical (TCM) system have not been elaborated. The two types of agarwood samples were evaluated in terms of trait characteristics, physicochemical indicators, key component groups, and global compositional profile. Furthermore, a molecular docking was performed to investigate the active ingredients. In vitro activity assays were performed to evaluate the activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) by GKA and normal agarwood. The results revealed that, overall, the traits, microscopic characteristics, chemical composition types, and bioactivity between GKA and normal agarwood were similar. The main differences were the content of resin (ethanolic extract content), the content of key component groups, and the composition of the different parent structural groups of 2-(2-phenethyl) chromones (PECs). The contents of total PEC and ethanol extract content of GKA were significantly higher than those of normal agarwood. The MS-based high-throughput analysis revealed that GKA has higher concentrations of sesquiterpenes and flindersia-type 2-(2-phenylethyl) chromones (FTPECs) (m/z 250-312) than normal agarwood. Molecular docking revealed that parent structural groups of FTPECs activated multiple signaling pathways, including the AMPK pathway, suggesting that FTPECs are major active components in GKA. The aim of this paper is to describe the intrinsic reasons for GKA as a high-quality agarwood and a potential source for novel drug development. We combined high-throughput mass spectrometry and multivariate statistical analysis to infer the different components of the two types of agarwood. Then we combined virtual screening and in vitro activity to construct a component/pharmacodynamic relationship to explore the causes of the activity differences between agarwood with different levels of quality and to identify potentially valuable lead compounds. This strategy can also be used for the comprehensive study of other TCMs with different qualities.

Establishment of a repertoire of fertility associated sperm proteins and their differential abundance in buffalo bulls (Bubalus bubalis) with contrasting fertility

Sperm harbours a wide range of proteins regulating their functions and fertility. In the present study, we made an effort to characterize and quantify the proteome of buffalo bull spermatozoa, and to identify fertility associated sperm proteins through comparative proteomics. Using high-throughput mass spectrometry platform, we identified 1305 proteins from buffalo spermatozoa and found that these proteins were mostly enriched in glycolytic process, mitochondrial respiratory chain, tricarboxylic acid cycle, protein folding, spermatogenesis, sperm motility and sperm binding to zona pellucida (p < 7.74E−08) besides metabolic (p = 4.42E−31) and reactive oxygen species (p = 1.81E−30) pathways. Differential proteomic analysis revealed that 844 proteins were commonly expressed in spermatozoa from both the groups while 77 and 52 proteins were exclusively expressed in high- and low-fertile bulls, respectively. In low-fertile bulls, 75 proteins were significantly (p < 0.05) upregulated and 176 proteins were significantly (p < 0.05) downregulated; these proteins were highly enriched in mitochondrial respiratory chain complex I assembly (p = 2.63E−07) and flagellated sperm motility (p = 7.02E−05) processes besides oxidative phosphorylation pathway (p = 6.61E−15). The down regulated proteins in low-fertile bulls were involved in sperm motility, metabolism, sperm-egg recognition and fertilization. These variations in the sperm proteome could be used as potential markers for the selection of buffalo bulls for fertility.

Dissecting molecular regulation of unconventional protein secretion.

Many elegant studies have revealed conserved molecular principles underlying the classical ER-Golgi-Secretory pathway for protein secretion. However, over the past two decades, several cytoplasmic proteins involved in innate immunity, angiogenesis, and neuronal pathogenicity have been shown to be secreted via ER-Golgi independent, “unconventional” secretory routes. Thus, understanding the molecular regulation of unconventional cytoplasmic protein secretion (UCPS) is of considerable interest. Our current studies are focused on 1) identifying unconventionally secreted proteins using an unbiased high-throughput mass-spectrometry based approach and 2) identifying regulators of UCPS using whole-genome CRISPR screening. By comparing levels of a given protein in conditioned media and cell lysates, our analysis effectively discriminates between authentically secreted proteins and non-secreted proteins released into the extracellular media from dead or dying cells. Coupling this approach with treatment of cells with Brefeldin-A, an inhibitor of ER-Golgi traffic, we have identified Brefeldin-A resistant peptides derived from candidate unconventionally secreted proteins from Huh7 (hepatocyte) and THP1 (immune) cell lines and primary endothelial cells. As expected, most highly secreted proteins from these cell lines were observed to contain signal peptides, presumably mediating traffic through the conventional ER/Golgi secretory pathway, with a minor fraction of the secretome consisting of unconventionally secreted proteins. Our analysis has identified several novel candidate UCPS cargoes, as well as confirming previously reported UCPS cargoes including metallothionein and leukocyte elastase inhibitor. A preliminary whole genome CRISPR screen for modulators of secretion for the known UCPS cargo, interleukin-1β (IL-1β), identified PIK3C2A (a lipid kinase known to be localized to the endo-lysosomal system), molecular motor proteins, and other protein kinases as candidate regulators of UCPS. Our preliminary studies also suggest significant heterogeneity in the regulation of UCPS across cell types, with PIK3C2A selectively influencing IL-1β secretion in non-pyroptotic cells.