Proteomic Methods Research Articles

A highly sensitive approach for the analysis of tyrosine phosphoproteome in primary T cells

Tyrosine phosphorylation, a common post-translational modification process for proteins, is involved in a variety of biological processes. However, the abundance of tyrosine-phosphorylated proteins is very low, making their identification by mass spectrometry (MS) is difficult; thus, milligrams of the starting material are often required for their enrichment. For example, tyrosine phosphorylation plays an important role in T cell signal transduction. However, the number of primary T cells derived from biological tissue samples is very small, and these cells are difficult to culture and expand; thus, the study of T cell signal transduction is usually carried out on immortalized cell lines, which can be greatly expanded. However, the data from immortalized cell lines cannot fully mimic the signal transduction processes observed in the real physiological state, and they usually lead to conclusions that are quite different from those of primary T cells. Therefore, a highly sensitive proteomic method was developed for studying tyrosine phosphorylation modification signals in primary T cells. To address the issue of the limited T cells numbers, a comprehensive protocol was first optimized for the isolation, activation, and expansion of primary T cells from mouse spleen. CD3+ primary T cells were successfully sorted; more than 91% of the T cells collected were well activated on day 2, and the number of T cells expanded to over 7-fold on day 4. Next, to address the low abundance of tyrosine-phosphorylated proteins, we used SH2-superbinder affinity enrichment and immobilized Ti4+affinity chromatography (Ti4+-IMAC) to enrich the tyrosine-phosphorylated polypeptides of primary T cells that were co-stimulated with anti-CD3 and anti-CD28. These polypeptides were resolved using nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Finally, 282 tyrosine phosphorylation sites were successfully identified in 1 mg of protein, including many tyrosine phosphorylation sites on the immunoreceptor tyrosine-based activation motif (ITAM) in the intracellular region of the T cell receptor membrane protein CD3, as well as the phosphotyrosine sites of ZAP70, LAT, VAV1, and other proteins related to signal transduction under costimulatory conditions. In summary, to solve the technical problems of the limited number of primary cells, low abundance of tyrosine-phosphorylated proteins, and difficulty of detection by MS, we developed a comprehensive proteomic method for the in-depth analysis of tyrosine phosphorylation modification signals in primary T cells. This protocol may be applied to map signal transduction networks that are closely related to physiological states.

Mechanism of azole resistance in Candida vulturna, an emerging multidrug resistant pathogen related with Candida haeumulonii and Candida auris.

Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents. This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna. We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created. Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype. This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance.

Analysis of the mechanism of fibrauretine alleviating Alzheimer's disease based on transcriptomics and proteomics.

The dried rattan stem of the Fibraurea Recisa Pierre plant contains the active ingredient known as fibrauretine (FN). Although it greatly affects Alzheimer's disease (AD), the mechanism of their effects still remains unclear. Proteomics and transcriptomics analysis methods were used in this study to determine the mechanism of FN in the treatment of AD. AD model is used through bilateral hippocampal injection of Aβ1-40. After successful modeling, FN was given for 30 days. The results showed that FN could improve the cognitive dysfunction of AD model rats, reduce the expression of Aβ and P-Tau, increase the content of acetylcholine and reduce the activity of acetylcholinesterase. The Kyoto Encyclopedia of Genes and Genomes enriched differentially expressed genes and proteins are involved in signaling pathways including metabolic pathway, AD, pathway in cancer, PI3K-AKT signaling pathway, and cAMP signaling pathway. Transcriptomics and proteomics sequencing resulted in 19 differentially expressed genes and proteins. Finally, in contrast to the model group, after FN treatment, the protein expressions and genes associated with the PI3K-AKT pathway were significantly improved in RT-qPCR and Western blot and assays. This is consistent with the findings of transcriptomic and proteomic analyses. Our study found that, FN may improve some symptoms of AD model rats through PI3K-AKT signaling pathway.

Delineating protein biomarkers for gastric cancers: A catalogue of mass spectrometry-based markers and assessment of their suitability for targeted proteomics applications

Gastric cancer (GC) is a global health concern. To facilitate improved management of GCs, protein biomarkers have been identified through mass spectrometry-based proteomics platforms. In order to exhibit clinical utility of such data, we congregated over 6800 differentially regulated proteins in GCs from proteomics studies and recorded the mass spectrometry platforms, association of the protein with infectious agents, protein identifiers, sample size and clinical characters of samples used with details on validation. Development of targeted proteomics methods is the cornerstone for pursuing these markers into clinical utility. Therefore, we developed Protein Biomarker Matrix for Gastric Cancer (PBMGC), a simple catalogue of robustness of each protein. This analysis yielded the identification of robust tissue, serum, and urine diagnostic and prognostic protein biomarker panels which can be further tested for their clinical utility. We also ascertained proteotypic tryptic peptides of 5631 proteins suitable for developing multiple reaction monitoring (MRM) assays. Extensive characterization of these peptides was carried out to record peptide ions, mass/charge and enhanced specific peptide features. With the vision of catering to proteomics researchers, the data generated through this analysis has been catalogued at Gastric Cancer Proteomics DataBase (GCPDB) (https://ciods.in/gcpdb/). Users can browse and download the data and improve GCPDB by submitting recently published data. SignificanceMass spectrometry-based proteomics platforms have accumulated substantial data on proteins differentially regulated in gastric cancer (GC) clinical samples. The utility of such data in clinical applications is limited by search for suitable biomarker panels for assessment of GCs.We assembled over 6800 differentially regulated proteins in GCs from proteomics studies and recorded the corresponding details including mass spectrometry platforms, status on the association of the protein with infectious agents, protein identifiers from different databases, sample size and clinical characters of samples used in test and control conditions along with details on their validation.Towards the vision of utilizing these markers in clinical assays, Protein Biomarker Matrix for Gastric Cancer (PBMGC) was developed and clinically relevant multi-protein panels were identified. We also demonstrated identification and characterization of tryptic proteotypic tryptic peptides of 5631 proteins biomarkers of GCs which are suitable for development of MRM assays in a SCIEX QTRAP instrument.Aimed to caterproteomics researchers, the data generated through this analysis has been catalogued at Gastric Cancer Proteomics DataBase (GCPDB) (https://ciods.in/gcpdb/). The users can browse and download details on different markers and improve GCPDB by submitting recently published data.Such an analysis could lay a cornerstone for building more such resources or conduct such analysis in different clinical conditions to uptake and develop targeted proteomics as the method of choice for clinical applications.

Physiological Adaptation of Fenneropenaeus chinensis in Response to Saline-Alkaline Stress Revealed by a Combined Proteomics and Metabolomics Method.

The rapid development of the mariculture industry has been hindered by limited coastal aquaculture space. To utilize the abundant inland saline-alkaline water, we studied the physiological effects of high carbonate alkalinity stress and high pH stress on Fenneropenaeus chinensis. The study employed quantitative proteomics by tandem mass tag (TMT) and non-targeted metabolomics analysis using a liquid chromatograph mass spectrometer (LC-MS) to understand the physiological and biochemical adaptive mechanisms of the hepatopancreas of F. chinensis in response to saline-alkaline stress at the molecular level. We designed two stress groups as follows: a high carbonate alkalinity (CA) group and a combined high carbonate alkalinity and high pH (CP) group. The study found that the protein and metabolic profiles of the two stress groups were changed, and the CP group, which was exposed to dual stresses, incurred more severe damage to the hepatopancreas compared to that of the CA group. After exposure to CA and CP, the hepatopancreas of F. chinensis showed significant alterations in 455 proteins and 50 metabolites, and 1988 proteins and 272 metabolites, respectively. In addition, F. chinensis upregulated the level of energy metabolism in the hepatopancreas to defend against osmotic imbalance caused by CA or CP stress, which was demonstrated by the significant upregulation of important proteins and metabolites in glycolysis, pyruvate metabolism, TCA cycle, and fatty acid oxidation. Additionally, pattern recognition receptors, the phenol oxidase system, and various immune-related metabolic enzymes and metabolites were also affected. The immune homeostasis of F. chinensis was affected by the alteration of the antioxidant system following exposure to CA or CP. These findings provide valuable information for F. chinensis saline-alkaline water cultivation practices.

A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract.

Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2-3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3-4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.

Proteomic Blueprint of Atlantic Cod (Gadus morhua) Otoliths Revealing Environmental Stress Insights through Label-Free Quantitative Shotgun Proteomics

Otoliths of the fish’s inner ear serve as a natural chronological recorder because of their continuous formation marked by daily, monthly, and annual increments. Despite their importance, the comprehensive protein content of otoliths remains not fully identified. Using the label-free shotgun proteomics method with one-dimensional liquid chromatography coupled to electrospray ionization-orbitrap tandem mass spectrometry, we quantified a broad range of proteins, with individual otoliths containing between 1341 and 1839 proteins. The identified proteins could potentially serve as a blueprint for fish growth from embryo to adult. We quantified eleven heat-shock proteins (HSPs) in both sexes and several proteins impacted by endocrine disruptors, indicating the otolith’s capacity to reflect environmental stress, potentially linked to climate change effects and altering of hormonal and neuroendocrine functions. Our bioinformatic ontology analysis confirmed the presence of proteins critical for various biological processes, including structural and enzymatic proteins. Protein–protein interaction (PPI) mapping also identified key interactions between the identified proteins. These findings significantly advance our understanding of otolith proteomics, offering a solid foundation for future work. Most of the identified proteins deposited daily and influenced by the environment were not implicated in the biomineralization of otolith, raising the potential for the otolith proteome to recreate details of fish life history at previously unrealized levels.

Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines.

Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases. While it is clear that cell lines derived from different cell types have differential proteome levels, increased understanding of large-scale functional differences requires additional information beyond abundance level measurements, including how protein conformations and interactions are altered in certain cell types to shape functional landscapes. Here, we employed quantitative in vivo protein cross-linking coupled to mass spectrometry to probe large-scale protein conformational and interaction changes among three commonly employed human cell lines, HEK293, MCF-7, and HeLa cells. Isobaric quantitative Protein Interaction Reporter (iqPIR) technologies were used to obtain quantitative values of cross-linked peptides across three cell lines. These data illustrated highly reproducible (R2 values larger than 0.8 for all biological replicates) quantitative interactome levels across multiple biological replicates. We also measured protein abundance levels in these cells using data independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteomics information allowed visualization of cell type-specific interactome changes mediated by proteome level adaptations as well as independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the biggest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study systems-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight on cellular functional landscapes.

Novel FFPE proteomics method suggests prolactin induced protein as hormone induced cytoskeleton remodeling spatial biomarker

Robotically assisted proteomics provides insights into the regulation of multiple proteins achieving excellent spatial resolution. However, developing an effective method for spatially resolved quantitative proteomics of formalin fixed paraffin embedded tissue (FFPE) in an accessible and economical manner remains challenging. We introduce non-robotic In-insert FFPE proteomics approach, combining glass insert FFPE tissue processing with spatial quantitative data-independent mass spectrometry (DIA). In-insert approach identifies 450 proteins from a 5 µm thick breast FFPE tissue voxel with 50 µm lateral dimensions covering several tens of cells. Furthermore, In-insert approach associated a keratin series and moesin (MOES) with prolactin-induced protein (PIP) indicating their prolactin and/or estrogen regulation. Our data suggest that PIP is a spatial biomarker for hormonally triggered cytoskeletal remodeling, potentially useful for screening hormonally affected hotspots in breast tissue. In-insert proteomics represents an alternative FFPE processing method, requiring minimal laboratory equipment and skills to generate spatial proteotype repositories from FFPE tissue.

Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry.

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this Review will serve as a handbook for researchers who are new to the field of bottom-up proteomics.

Covalent adduct formation of histone with organophosphorus pesticides in vitro

It is established that organophosphorus pesticide (OPP) toxicity results from modification of amino acids in active sites of target proteins. OPPs can also modify unrelated target proteins such as histones and such covalent histone modifications can alter DNA-binding properties and lead to aberrant gene expression. In the present study, we report on non-enzymatic covalent modifications of calf thymus histones adducted to selected OPPs and organophosphate flame retardants (OPFRs) in vitro using a bottom-up proteomics method approach. Histones were not found to form detectable adducts with the two tested OPFRs but were avidly modified by a few of the seven OPPs that were tested in vitro. Dimethyl phosphate (or diethyl phosphate) adducts were identified on Tyr, Lys and Ser residues. Most of the dialkyl phosphate adducts were identified on Tyr residues. Methyl and ethyl modified histones were also detected. Eleven amino residues in histones showed non-enzymatic covalent methylation by exposure of dichlorvos and malathion. Our bottom-up proteomics approach showing histone-OPP adduct formation warrants future studies on the underlying mechanism of chronic illness from exposure to OPPs.

High-throughput Proteomics-Guided Biomarker Discovery of Hepatocellular Carcinoma

Liver cancer stands as the fifth leading cause of cancer-related deaths globally. Hepatocellular carcinoma (HCC) comprises approximately 85%-90% of all primary liver malignancies. However, only 20-30% of HCC patients qualify for curative therapy, primarily due to the absence of reliable tools for early detection and prognosis of HCC. This underscores the critical need for molecular biomarkers for HCC management. Since proteins reflect disease status directly, proteomics has been utilized in biomarker developments for HCC. In particular, proteomics coupled with liquid chromatography-mass spectrometer (LC-MS) methods facilitate the process of discovering biomarker candidates for diagnosis, prognosis, and therapeutic strategies.In this work, we investigated LC-MS-based proteomics methods through recent reference reviews, with a particular focus on sample preparation and LC-MS methods appropriate for the discovery of HCC biomarkers and their clinical applications.We classified proteomics studies of HCC according to sample types, and we examined the coverage of protein biomarker candidates based on LC-MS methods in relation to study scales and goals. Comprehensively, we proposed protein biomarker candidates categorized by sample types and biomarker types for appropriate clinical use.In this review, we summarized recent LC-MS-based proteomics studies on HCC and proposed potential protein biomarkers. Our findings are expected to expand the understanding of HCC pathogenesis and enhance the efficiency of HCC diagnosis and prognosis, thereby contributing to improved patient outcomes.

Advancing drug development by leveraging microdissection for proteogenomic analysis of histological sections.

e14660 Background: Combining laser capture microdissection (LCM) with proteogenomics in cancer research offers a targeted way to way to explore the intricate interactions between the tumor microenvironment and tumor cells proper. For immuno-oncology (IO) research, this is especially important, since improvements in the predictability of patient response to IO-based drugs is greatly needed. An improved understanding of the spatial relationships involving tumor cells, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: From fresh frozen tissue, LCM is used to isolate and obtain distinct histological cell types. Nucleic acids and proteins are extracted from captured cells for in-depth multi-modality molecular profiling assays. This includes multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing the analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, DNA sequencing can be performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. RNA-seq may be performed for gene expression. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. Our novel proteogenomics approach is focused around a customized enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: A proteogenomic approach is reported that is utilizing amount limited tissue quantities from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Data Independent Acquisition to Inform the Development of Targeted Proteomics Assays Using a Triple Quadrupole Mass Spectrometer.

Mass spectrometry based targeted proteomics methods provide sensitive and high-throughput analysis of selected proteins. To develop a targeted bottom-up proteomics assay, peptides must be evaluated as proxies for the measurement of a protein or proteoform in a biological matrix. Candidate peptide selection typically relies on predetermined biochemical properties, data from semi-stochastic sampling, or by empirical measurements. These strategies require extensive testing and method refinement due to the difficulties associated with prediction of peptide response in the biological matrix of interest. Gas-phase fractionated (GPF) narrow window data-independent acquisition (DIA) aids in the development of reproducible selected reaction monitoring (SRM) assays by providing matrix-specific information on peptide detectability and quantification by mass spectrometry. To demonstrate the suitability of DIA data for selecting peptide targets, we reimplement a portion of an existing assay to measure 98 Alzheimer's disease proteins in cerebrospinal fluid (CSF). Peptides were selected from GPF-DIA based on signal intensity and reproducibility. The resulting SRM assay exhibits similar quantitative precision to published data, despite the inclusion of different peptides between the assays. This workflow enables development of new assays without additional up-front data acquisition, demonstrated here through generation of a separate assay for an unrelated set of proteins in CSF from the same dataset.

Transporter Protein Expression of Corneal Epithelium in Rabbit and Porcine: Evaluation of Models for Ocular Drug Transport Study.

The transcorneal route is the main entry route for drugs to the intraocular parts, after topical administration. The outer surface, the corneal epithelium (CE), forms the rate-limiting barrier for drug permeability. Information about the role and protein expression of drug and amino acid transporter proteins in the CE is sparse and lacking. The aim of our study was to characterize transporter protein expression in rabbit and porcine CE to better understand potential drug and nutrient absorption after topical administration. Proteins, mainly Abc and Slc transporters, were characterized with quantitative targeted absolute proteomics and global untargeted proteomics methods. In the rabbit CE, 24 of 48 proteins were detected in the targeted approach, and 21 of these were quantified. In the porcine CE, 26 of 58 proteins were detected in the targeted approach, and 20 of these were quantified. Among these, 15 proteins were quantified in both animals: 4f2hc (Slc3a2), Aqp0, Asct1 (Slc1a4), Asct2 (Slc1a5), Glut1 (Slc2a1), Hmit (Slc2a13), Insr, Lat1 (Slc7a5), Mct1 (Slc16a1), Mct2 (Slc16a7), Mct4 (Slc16a3), Mrp 4 (Abcc4), Na+/K+-ATPase, Oatp3a1 (Slco3a1), and Snat2 (Slc38a2). Overall, the global proteomics results supported the targeted proteomics results. Organic anion transporting polypeptide Oatp3a1 was detected and quantified for the first time in both rabbit (1.4 ± 0.4 fmol/cm2) and porcine (11.1 ± 5.3 fmol/cm2) CE. High expression levels were observed for L-type amino acid transporter, Lat1, which was quantified with newly selected extracellular domain peptides in rabbit (48.9 ± 11.8 fmol/cm2) and porcine (37.6 ± 11.5 fmol/cm2) CE. The knowledge of transporter protein expression in ocular barriers is a key factor in the successful design of new ocular drugs, pharmacokinetic modeling, understanding ocular diseases, and the translation to human.

ITRAQ-based proteomics revealed the acaricidal mechanism of methyl isoeugenol against Dermatophagoides farinae

ABSTRACT Dermatophagoides farinae is a major source of allergens that are the cause of allergic diseases in humans. Methyl isoeugenol (MIE) is a naturally occurring compound in plants and its strong acaricidal toxicity has been established; however, the mechanism behind its acaricidal properties remains unclear. In this study, to investigate the action mechanism of MIE on D. farinae the isobaric tags for relative and absolute quantification (iTRAQ) proteomics method is used. Protein samples were extracted from D. farinae before and after 24 h of MIE treatment for iTRAQ technique analysis. Bioinformatics analysis revealed that most differentially expressed proteins were located in the cytoplasm, impacting cytoplasmic function. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed significant enrichment of the differential proteins in the cellular autophagy signalling pathway. The autophagy-related proteins Autophagy-Related Protein 3 (ATG3) and Cathepsin L (CTSL) were up-regulated, disrupting the autophagic process. We found that the main action mechanism of MIE against D. farinae might be cellular autophagy. The transcriptional validation of six differential proteins demonstrated consistent expression trends with the proteomics data. This study reveals the acaricidal mechanism of MIE for the first time and lays the foundation for further research on D. farinae control.

Spatially resolved distribution of pancreatic hormones proteoforms by MALDI-imaging mass spectrometry

Zinc plays a crucial role both in the immune system and endocrine processes. Zinc restriction in the diet has been shown to lead to degeneration of the endocrine pancreas, resulting in hormonal imbalance within the β-cells. Proteostasismay vary depending on the stage of a pathophysiological process, which underscores the need for tools aimed at directly analyzing biological status. Among proteomics methods, MALDI-ToF-MS can serve as a rapid peptidomics tool for analyzing extracts or by histological imaging. Here we report the optimization of MALDI imaging mass spectrometry analysis of histological thin sections from mouse pancreas. This optimization enables the identification of the major islet peptide hormones as well as the major accumulated precursors and/or proteolytic products of peptide hormones. Cross-validation of the identified peptide hormones was performed by LC-ESI-MS from pancreatic islet extracts. Mice subjected to a zinc-restricted diet exhibited a relatively lower amount of peptide intermediates compared to the control group. These findings provide evidence for a complex modulation of proteostasis by micronutrients imbalance, a phenomenon directly accessed by MALDI-MSI.

Proteomic assay for rapid characterisation of Staphylococcus aureus antimicrobial resistance mechanisms directly from blood cultures.

Staphylococcus aureus is one of the most common pathogens causing bloodstream infection. A rapid characterisation of resistance to methicillin and, occasionally, to aminoglycosides for particular indications, is therefore crucial to quickly adapt the treatment and improve the clinical outcomes of septic patients. Among analytical technologies, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a promising tool to detect resistance mechanisms in clinical samples. A rapid proteomic method was developed to detect and quantify the most clinically relevant antimicrobial resistance effectors in S. aureus in the context of sepsis: PBP2a, PBP2c, APH(3')-III, ANT(4')-I, and AAC(6')-APH(2''), directly from positive blood cultures and in less than 70min including a 30-min cefoxitin-induction step. The method was tested on spiked blood culture bottles inoculated with 124 S.aureus, accounting for the known genomic diversity of SCCmec types and the genetic background of the strains. This method provided 99% agreement for PBP2a (n = 98/99 strains) detection. Agreement was 100% for PBP2c (n = 5/5), APH(3')-III (n = 16/16), and ANT(4')-I (n = 20/20), and 94% for AAC(6')-APH(2'') (n = 16/17). Across the entire strain collection, 100% negative agreement was reported for each of the 5 resistance proteins. Additionally, relative quantification of ANT(4')-I expression allowed to discriminate kanamycin-susceptible and -resistant strains, in all strains harbouring the ant(4')-Ia gene. The LC-MS/MS method presented herein demonstrates its ability to provide a reliable determination of S. aureus resistance mechanisms, directly from positive blood cultures and in a short turnaround time, as required in clinical laboratories.

On the utility of ultrafast MS1-only proteomics in drug target discovery studies based on thermal proteome profiling method.

Advances in high-throughput high-resolution mass spectrometry and the development of thermal proteome profiling approach (TPP) have made it possible to accelerate a drug target search. Since its introduction in 2014, TPP quickly became a method of choice in chemical proteomics for identifying drug-to-protein interactions on a proteome-wide scale and mapping the pathways of these interactions, thus further elucidating the unknown mechanisms of action of a drug under study. However, the current TPP implementations based on tandem mass spectrometry (MS/MS), associated with employing lengthy peptide separation protocols and expensive labeling techniques for sample multiplexing, limit the scaling of this approach for the ever growing variety of drug-to-proteomes. A variety of ultrafast proteomics methods have been developed in the last couple of years. Among them, DirectMS1 provides MS/MS-free quantitative proteome-wide analysis in 5-min time scale, thus opening the way for sample-hungry applications, such as TPP. In this work, we demonstrate the first implementation of the TPP approach using the ultrafast proteome-wide analysis based on DirectMS1. Using a drug topotecan, which is a known topoisomerase I (TOP1) inhibitor, the feasibility of the method for identifying drug targets at the whole proteome level was demonstrated for an ovarian cancer cell line.

Comprehensive analysis of silk proteins and gland compartments in Limnephilus lunatus, a case-making trichopteran

Caddisfly larvae produce silk containing heavy and light fibroins, similar to the silk of Lepidoptera, for the construction of underwater structures. We analyzed the silk of Limnephilus lunatus belonging to the case-forming suborder Integripalpia. We analyzed the transcriptome, mapped the transcripts to a reference genome and identified over 80 proteins using proteomic methods, and checked the specificity of their expression. For comparison, we also analyzed the transcriptome and silk proteome of Limnephilus flavicornis. Our results show that fibroins and adhesives are produced together in the middle and posterior parts of the silk glands, while the anterior part produces enzymes and an unknown protein AT24. The number of silk proteins of L. lunatus far exceeds that of the web-spinning Plectrocnemia conspersa, a previously described species from the suborder Annulipalpia. Our results support the idea of increasing the structural complexity of silk in rigid case builders compared to trap web builders.