RESUMEN

Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.

Asunto(s)

Inmunoprecipitación , Fosfoproteínas , Espectrometría de Masas en Tándem , Fosforilación , Espectrometría de Masas en Tándem/métodos , Inmunoprecipitación/métodos , Cromatografía Liquida/métodos , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Espectrometría de Masas/métodos

RESUMEN

Protein S-sulfhydration involves the regulation of various protein functions, and resolving the S-sulfhydrated proteome (persulfidome) allows for a deeper exploration of various redox regulations. Therefore, we designed a reducible covalent capture method for isolating S-sulfhydrated proteins, which can analyze the persulfidome in biological samples and monitor specific S-sulfhydrated proteins. In this study, we applied this method to reveal the S-sulfhydration levels of proteins, including 3-phosphoglyceraldehyde dehydrogenase, NFκB/p65, and nucleolin. Furthermore, this technique can be used to enrich S-sulfhydrated peptides, aiding in the determination of protein S-sulfhydration modification sites. Finally, we observed that the S-sulfhydration and oxidation of nucleolin on the C543 residue correlate with its nuclear translocation, downstream regulation of p53, Bcl-xL, and Bcl-2 RNA levels and protein expression, as well as the protective function against oxidative stress. Therefore, this method may facilitate the understanding of the regulation of protein function by redox perturbation.

Asunto(s)

Nucleolina , Oxidación-Reducción , Fosfoproteínas , Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/análisis , Humanos , Proteoma/análisis , Proteoma/química

RESUMEN

This study aimed to reveal the change patterns of the phosphorylation modification status of yak whey phosphoproteins during lactation and their physiological effects. Herein, we comprehensively characterized whey phosphoproteome in yak colostrum and mature milk using an ultra-high throughput phosphoproteomics approach incorporating trapped ion mobility technology. A total of 344 phosphorylation sites from 206 phosphoproteins were identified, with individual site modification predominating. Notably, 117 significantly different phosphorylation sites were distributed on 89 whey phosphoproteins. Gene ontology analysis indicated that these significantly different whey phosphoproteins (SDWPPs) were mainly annotated to carbohydrate metabolic process, membrane, extracellular region and calcium ion binding. Metabolic pathway enrichment analysis demonstrated that SDWPPs were critically involved in protein processing in endoplasmic reticulum, NOD-like receptor signaling pathway and N-glycan biosynthesis. Our results elucidate the phosphorylation profiles of yak whey phosphoproteins at different lactations and their adaptive regulatory role in meeting the nutritional requirements of yak calves during development.

Asunto(s)

Calostro , Leche , Fosfoproteínas , Proteómica , Proteína de Suero de Leche , Animales , Bovinos/metabolismo , Proteína de Suero de Leche/metabolismo , Proteína de Suero de Leche/química , Calostro/química , Calostro/metabolismo , Leche/química , Leche/metabolismo , Femenino , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Fosforilación , Lactancia

RESUMEN

Quantitative phosphoproteomic data has mostly been reported from experiments comparing relative phosphopeptides intensities in two or more different conditions, while the ideal parameter to compare is phosphopeptides occupancies. This term is scarcely used and therefore barely implemented in phosphoproteomics studies, and this should be of concern for the scientific journals. In order to demonstrate the relevance of this issue, here we show how the method of choice affects the interpretation of the data. The phosphoproteomic profile modulated in two AML cell lines after CK2 inhibition with CIGB-300 or CX-4945 is shown. Following the downstream action of CK2 the phosphosite intensity and occupancy results were compared to validate the best approach for quantitative phosphoproteomic studies. Even when the total number of quantified phosphopeptides was higher by using the intensity calculation, in all the cases the percent of CK2 consensus sequences which were down-regulated in response to CK2 inhibition was higher using the phosphosite occupancy quantification. To note, a high number of CK2 consensus sequences was found down-regulated with at least a 10% or 15% of phosphosite occupancy variation illustrating that low thresholds of occupancy modulation might be indicative of biological effect. Additionally, several biological processes only appear significantly over-represented in the phosphoproteome quantified by occupancy. The functional enrichment analysis per ranges of occupancy variations also illustrated clear differences among AML cell lines subjected to CK2 inhibition by CX-4945. A low overlap between the phosphoproteomes quantified by intensity and occupancy was obtained illustrating that new developments in proteomics techniques are needed to improve the performance of the occupancy approach. Even in such context, results indicate that occupancy quantification performs better than phosphorylation quantification based on intensity reinforcing the importance of such quantification approach to describe phosphoproteomic data.

Asunto(s)

Quinasa de la Caseína II , Fosfopéptidos , Proteómica , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/antagonistas & inhibidores , Humanos , Fosfopéptidos/análisis , Fosfopéptidos/metabolismo , Proteómica/métodos , Línea Celular Tumoral , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Fosforilación , Naftiridinas/farmacología , Fenazinas , Proteoma/análisis , Proteoma/metabolismo , Leucemia Mieloide Aguda/metabolismo

RESUMEN

Formalin-fixed paraffin-embedded (FFPE) tissues are suitable for proteomic and phosphoproteomic biomarker studies by data-independent acquisition mass spectrometry. The choice of the sample preparation method influences the number, intensity, and reproducibility of identifications. By comparing four deparaffinization and rehydration methods, including heptane, histolene, SubX, and xylene, we found that heptane and methanol produced the lowest coefficients of variation (CVs). Using this, five extraction methods from the literature were modified and evaluated for their performance using kidney, leg muscle, lung, and testicular rat organs. All methods performed well, except for SP3 due to insufficient tissue lysis. Heat n' Beat was the fastest and most reproducible method with the highest digestion efficiency and lowest CVs. S-Trap produced the highest peptide yield, while TFE produced the best phosphopeptide enrichment efficiency. The quantitation of FFPE-derived peptides remains an ongoing challenge with bias in UV and fluorescence assays across methods, most notably in SPEED. Functional enrichment analysis demonstrated that each method favored extracting some gene ontology cellular components over others including chromosome, cytoplasmic, cytoskeleton, endoplasmic reticulum, membrane, mitochondrion, and nucleoplasm protein groups. The outcome is a set of recommendations for choosing the most appropriate method for different settings.

Asunto(s)

Adhesión en Parafina , Proteómica , Proteómica/métodos , Animales , Ratas , Formaldehído/química , Masculino , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Fosfoproteínas/aislamiento & purificación , Fijación del Tejido , Riñón/metabolismo , Riñón/química

RESUMEN

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology.

Asunto(s)

Espectrometría de Masas , Fosfoproteínas , Proteoma , Proteómica , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Animales , Células HeLa , Fosforilación , Ratones , Proteoma/metabolismo , Espectrometría de Masas/métodos , Proteómica/métodos

RESUMEN

Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl ß-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 µg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.

Asunto(s)

Fosfopéptidos , Fosfoproteínas , Proteómica , Flujo de Trabajo , Proteómica/métodos , Humanos , Fosfopéptidos/análisis , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/química , Reproducibilidad de los Resultados , Receptores ErbB/metabolismo , Línea Celular Tumoral , Fosforilación , Titanio/química , Neoplasias Pulmonares/metabolismo , Espectrometría de Masas/métodos

RESUMEN

Analyzing the phosphoproteome at nanoscale poses a significant challenge, mainly due to the substantial sample loss from nonspecific surface adsorption during the enrichment of low stoichiometric phosphopeptides. Here, we describe a tandem tip-based phosphoproteomics sample preparation method capable of sequential sample cleanup and enrichment without the need for additional sample transfer, thereby minimizing sample loss. Integration of this method to our recently developed SOP (surfactant-assisted one-pot sample preparation) and iBASIL (improved boosting to amplify signal with isobaric labeling) approaches creates a streamlined workflow, enabling sensitive, high-throughput nanoscale phosphoproteomics measurements.

Asunto(s)

Fosfopéptidos , Fosfoproteínas , Proteómica , Flujo de Trabajo , Proteómica/métodos , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Fosfopéptidos/análisis , Humanos , Espectrometría de Masas en Tándem/métodos

RESUMEN

The rapid epidemic around the world of coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, proves the need and stimulates efforts to explore efficient diagnostic tests for the sensitive detection of the SARS-CoV-2 virus. An aggregation-induced electrochemiluminescence (AIECL) sensor was developed for the ultrasensitive detection of the SARS-CoV-2 nucleocapsid (N) protein in this work. Tetraphenylethylene doped in zeolite imidazole backbone-90 (TPE-ZIF-90) showed highly efficient aggregation-induced emission (AIE) to endow TPE-ZIF-90 with high ECL intensity. Upon the capture of the SARS-CoV-2 N protein by immune recognition, an alkaline phosphatase (ALP)-modified gold nanoparticle (AuNP)-decorated zinc oxide (ZnO) nanoflower (ALP/Au-ZnO) composite was introduced on the sensing platform, which catalyzed L-ascorbate-2-phosphate trisodium salt (AA2P) to produce PO43- and ascorbic acid (AA). Based on a multiquenching of the ECL signal strategy, including resonance energy transfer (RET) between TPE-ZIF-90 and Au-ZnO, disassembly of TPE-ZIF-90 triggered by the strong coordination between PO43- and Zn2+, and RET between TPE-ZIF-90 and AuNPs produced in situ by the AA reductive reaction, the constructed AIECL sensor achieved highly sensitive detection of the SARS-CoV-2 N protein with a low limit of detection of 0.52 fg/mL. With the merits of high specificity, good stability, and proven application ability, the present RET- and enzyme-triggered multiquenching AIECL sensor may become a powerful tool in the field of SARS-CoV-2 virus diagnosis.

Asunto(s)

Técnicas Electroquímicas , Oro , Mediciones Luminiscentes , Nanopartículas del Metal , SARS-CoV-2 , Óxido de Zinc , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Nanopartículas del Metal/química , Oro/química , Técnicas Electroquímicas/métodos , Mediciones Luminiscentes/métodos , Humanos , Óxido de Zinc/química , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/análisis , Límite de Detección , COVID-19/diagnóstico , COVID-19/virología , Técnicas Biosensibles/métodos , Fosfoproteínas/análisis , Fosfoproteínas/química , Fosfoproteínas/inmunología , Estilbenos/química , Zeolitas/química , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/química , Imidazoles/química

RESUMEN

An enhanced lateral flow assay (LFA) is presented for rapid and highly sensitive detection of acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens with gold nanoflowers (Au NFs) as signaling markers and gold enhancement to amplify the signal intensities. First, the effect of the morphology of gold nanomaterials on the sensitivity of LFA detection was investigated. The results showed that Au NFs prepared by the seed growth method showed a 5-fold higher detection sensitivity than gold nanoparticles (Au NPs) of the same particle size, which may benefit from the higher extinction coefficient and larger specific surface area of Au NFs. Under the optimized experimental conditions, the Au NFs-based LFA exhibited a detection limit (LOD) of 25 pg mL-1 for N protein using 135 nm Au NFs as the signaling probes. The signal was further amplified by using a gold enhancement strategy, and the LOD for the detection of N protein achieved was 5 pg mL-1. The established LFA also exhibited good repeatability and stability and showed applicability in the diagnosis of SARS-CoV-2 infection.

Asunto(s)

Antígenos Virales , Proteínas de la Nucleocápside de Coronavirus , Oro , Límite de Detección , Nanopartículas del Metal , SARS-CoV-2 , Oro/química , SARS-CoV-2/inmunología , Nanopartículas del Metal/química , Humanos , Antígenos Virales/análisis , Antígenos Virales/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/análisis , Fosfoproteínas/inmunología , Fosfoproteínas/análisis , Fosfoproteínas/química , COVID-19/diagnóstico , COVID-19/virología , Inmunoensayo/métodos , Prueba Serológica para COVID-19/métodos

RESUMEN

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.

Asunto(s)

Fosfoproteínas , Proteoma , Linfocitos T , Tirosina , Animales , Ratones , Fosforilación , Fosfoproteínas/análisis , Proteoma/análisis , Proteómica/métodos , Transducción de Señal

RESUMEN

The sensitivity of phosphorylation site identification by mass spectrometry (MS)-based phosphoproteomics has improved significantly. However, the lack of kinase-substrate relationship (KSR) data has hindered improvement of the range and accuracy of kinase activity prediction using phosphoproteome data. We herein describe the application of a systematic identification of KSR by integrated phosphoproteome and interactome analysis using doxycycline (Dox)-induced target kinase-overexpressing HEK-293 cells.

Asunto(s)

Fosfoproteínas , Proteoma , Proteómica , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Células HEK293 , Proteómica/métodos , Fosforilación , Proteoma/metabolismo , Especificidad por Sustrato , Espectrometría de Masas/métodos , Proteínas Quinasas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Doxiciclina/farmacología

RESUMEN

The spread of the SARS-CoV-2 virus has had an unprecedented impact, both by posing a serious risk to human health and by amplifying the burden on the global economy. The rapid identification of the SARS-CoV-2 virus has been crucial to preventing and controlling the spread of SARS-CoV-2 infections. In this study, we propose a multilayered plasmonic nanotrap (MPNT) device for the rapid identification of single particles of SARS-CoV-2 virus in ultra-high sensitivity by surface-enhanced Raman scattering (SERS). The MPNT device is composed of arrays of concentric cylindrical cavities with Ag/SiO2/Ag multilayers deposited on the top and at the bottom. By varying the diameter of the cylinders and the thickness of the multilayers, the resonant optical absorption and local electric field were optimized. The SERS enhancement factors of the proposed device are of the order of 108, which enable the rapid identification of SARS-CoV-2 N protein in concentrations as low as 1.25 × 10-15－12.5 × 10-15 g mL-1 within 1 min. The developed MPNT SERS device provides a label-free and rapid detection platform for SARS-CoV-2 virus. The general nature of the device makes it equally suitable to detect other infectious viruses.

Asunto(s)

COVID-19 , SARS-CoV-2 , Plata , Espectrometría Raman , Espectrometría Raman/métodos , SARS-CoV-2/aislamiento & purificación , Plata/química , Humanos , COVID-19/diagnóstico , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/análisis , Dióxido de Silicio/química , Fosfoproteínas/análisis , Fosfoproteínas/química , Nanopartículas del Metal/química , Límite de Detección , Proteínas de la Nucleocápside/química

RESUMEN

SUMMARY: Identification and quantification of phosphorylation sites are essential for biological interpretation of a phosphoproteomics experiment. For data independent acquisition mass spectrometry-based (DIA-MS) phosphoproteomics, extracting a site-level report from the output of current processing software is not straightforward as multiple peptides might contribute to a single site, multiple phosphorylation sites can occur on the same peptides, and protein isoforms complicate site specification. Currently only limited support is available from a commercial software package via a platform-specific solution with a rather simple site quantification method. Here, we present sitereport, a software tool implemented in an extendable Python package called msproteomics to report phosphosites and phosphopeptides from a DIA-MS phosphoproteomics experiment with a proven quantification method called MaxLFQ. We demonstrate the use of sitereport for downstream data analysis at site level, allowing benchmarking different DIA-MS processing software tools. AVAILABILITY AND IMPLEMENTATION: sitereport is available as a command line tool in the Python package msproteomics, released under the Apache License 2.0 and available from the Python Package Index (PyPI) at https://pypi.org/project/msproteomics and GitHub at https://github.com/tvpham/msproteomics.

Asunto(s)

Fosfoproteínas , Proteómica , Programas Informáticos , Proteómica/métodos , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Espectrometría de Masas/métodos , Fosforilación , Fosfopéptidos/análisis , Fosfopéptidos/metabolismo

RESUMEN

BACKGROUND: Kinases are intracellular signalling mediators and key to sustaining the inflammatory process in rheumatoid arthritis (RA). Oral inhibitors of Janus Kinase family (JAKs) are widely used in RA, while inhibitors of other kinase families e.g. phosphoinositide 3-kinase (PI3K) are under development. Most current biomarker platforms quantify mRNA/protein levels, but give no direct information on whether proteins are active/inactive. Phosphoproteome analysis has the potential to measure specific enzyme activation status at tissue level. METHODS: We validated the feasibility of phosphoproteome and total proteome analysis on 8 pre-treatment synovial biopsies from treatment-naive RA patients using label-free mass spectrometry, to identify active cell signalling pathways in synovial tissue which might explain failure to respond to RA therapeutics. RESULTS: Differential expression analysis and functional enrichment revealed clear separation of phosphoproteome and proteome profiles between lymphoid and myeloid RA pathotypes. Abundance of specific phosphosites was associated with the degree of inflammatory state. The lymphoid pathotype was enriched with lymphoproliferative signalling phosphosites, including Mammalian Target Of Rapamycin (MTOR) signalling, whereas the myeloid pathotype was associated with Mitogen-Activated Protein Kinase (MAPK) and CDK mediated signalling. This analysis also highlighted novel kinases not previously linked to RA, such as Protein Kinase, DNA-Activated, Catalytic Subunit (PRKDC) in the myeloid pathotype. Several phosphosites correlated with clinical features, such as Disease-Activity-Score (DAS)-28, suggesting that phosphosite analysis has potential for identifying novel biomarkers at tissue-level of disease severity and prognosis. CONCLUSIONS: Specific phosphoproteome/proteome signatures delineate RA pathotypes and may have clinical utility for stratifying patients for personalised medicine in RA.

Asunto(s)

Artritis Reumatoide , Fosfoproteínas , Proteómica , Transducción de Señal , Membrana Sinovial , Humanos , Artritis Reumatoide/metabolismo , Membrana Sinovial/metabolismo , Transducción de Señal/fisiología , Proteómica/métodos , Femenino , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Persona de Mediana Edad , Masculino , Adulto , Anciano , Proteoma/análisis , Proteoma/metabolismo

RESUMEN

This paper reports a rapid and sensitive sensor for the detection and quantification of the COVID-19 N-protein (N-PROT) via an electrochemical mechanism. Single-frequency electrochemical impedance spectroscopy was used as a transduction method for real-time measurement of the N-PROT in an immunosensor system based on gold-conjugate-modified carbon screen-printed electrodes (Cov-Ag-SPE). The system presents high selectivity attained through an optimal stimulation signal composed of a 0.0 V DC potential and 10 mV RMS-1 AC signal at 100 Hz over 300 s. The Cov-Ag-SPE showed a log response toward N-PROT detection at concentrations from 1.0 ng mL-1 to 10.0 µg mL-1, with a 0.977 correlation coefficient for the phase (θ) variation. An ML-based approach could be created using some aspects observed from the positive and negative samples; hence, it was possible to classify 252 samples, reaching 83.0, 96.2 and 91.3% sensitivity, specificity, and accuracy, respectively, with confidence intervals (CI) ranging from 73.0 to 100.0%. Because impedance spectroscopy measurements can be performed with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing, even in places with limited resources, as an alternative to the common diagnostics methods.

Asunto(s)

Técnicas Biosensibles , COVID-19 , Espectroscopía Dieléctrica , Oro , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/virología , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , Humanos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/inmunología , Espectroscopía Dieléctrica/instrumentación , Espectroscopía Dieléctrica/métodos , Oro/química , Electrodos , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Inmunoensayo/métodos , Inmunoensayo/instrumentación , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/análisis , Carbono/química , Fosfoproteínas/análisis

RESUMEN

Coronavirus disease 2019 (COVID-19) is a highly contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in a global health crisis. The primary diagnostic method for COVID-19 is quantitative reverse transcription PCR, which is time-consuming and requires expensive instrumentation. Here, we developed an electrochemical biosensor for detecting SARS-CoV-2 biomarkers using a 3D porous polyacrylamide/polyaniline hydrogel (PPG) electrode prepared by UV photopolymerization and in situ polymerization. The electrochemical immunosensor for detecting SARS-CoV-2 N protein via the immune sandwich principle demonstrated a lower detection limit of 42 pg/mL and comparable specificity to a commercial enzyme-linked immunosorbent assay, which was additionally validated in pseudoviruses. The electrochemical sensor for hydrogen peroxide showed a low detection limit of 0.5 µM and excellent selectivity, which was further confirmed in cancer cells under oxidative stress. The biomarkers of SARS-CoV-2 were successfully detected due to the signal amplification capability provided by 3D porous electrodes and the high sensitivity of the antigen-antibody specific binding. This study introduces a novel three-dimensional electrode with great potential for the early detection of SARS-CoV-2.

Asunto(s)

Técnicas Biosensibles , COVID-19 , Técnicas Electroquímicas , Electrodos , Hidrogeles , Peróxido de Hidrógeno , Límite de Detección , SARS-CoV-2 , Peróxido de Hidrógeno/química , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/inmunología , Humanos , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , COVID-19/diagnóstico , COVID-19/virología , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Hidrogeles/química , Proteínas de la Nucleocápside de Coronavirus/análisis , Proteínas de la Nucleocápside de Coronavirus/inmunología , Fosfoproteínas/análisis , Inmunoensayo/instrumentación , Inmunoensayo/métodos

RESUMEN

Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6 h (PI6, the early invasion phase) or 36 h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.

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Title: Analyse phosphoprotéomique quantitative de cellules DF-1 de poulet infectées par Eimeria tenella, par spectrométrie de masse avec marqueur de masse en tandem (TMT) et surveillance des réactions parallèles (PRM). Abstract: Eimeria tenella est un parasite intracellulaire obligatoire qui cause de graves dommages à l'industrie de l'élevage de volailles. La phosphorylation des protéines joue un rôle essentiel dans les interactions entre la cellule hôte et E. tenella. Cependant, aucune analyse phosphoprotéomique complète des cellules hôtes à différentes phases de l'infection par E. tenella n'a été publiée. Dans cette étude, une analyse phosphoprotéomique quantitative de fibroblastes DF-1 d'embryon de poulet non infectés (NI) ou infectés par E. tenella pendant 6 h (PI6, la phase d'invasion précoce) ou 36 h (PI36, la phase de développement des trophozoïtes) a été réalisée. Un total de 10 122 phosphopeptides correspondant à 3 398 phosphoprotéines de cellules hôtes ont été identifiés et 13 437 sites de phosphorylation ont été identifiés. Parmi celles-ci, 491, 1 253 et 275 protéines différentiellement phosphorylées exprimées ont été identifiées respectivement dans les comparaisons PI6/NI, PI36/NI et PI36/PI6. L'analyse d'enrichissement de la voie KEGG a montré qu'E. tenella modulait les processus de la cellule hôte par phosphorylation, y compris l'adhésion focale, la régulation du cytosquelette d'actine et la signalisation FoxO, pour aider sa phase d'invasion précoce, et la modulation des jonctions adhérentes et de la voie de signalisation ErbB pour favoriser le développement de son trophozoïte. Ces résultats enrichissent les données sur l'interaction entre E. tenella et les cellules hôtes et facilitent une meilleure compréhension des mécanismes moléculaires sous-jacents aux relations hôtes­parasites.

Asunto(s)

Pollos , Eimeria tenella , Fibroblastos , Fosfoproteínas , Proteómica , Espectrometría de Masas en Tándem , Animales , Eimeria tenella/fisiología , Pollos/parasitología , Proteómica/métodos , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Fosforilación , Fibroblastos/parasitología , Línea Celular , Enfermedades de las Aves de Corral/parasitología , Interacciones Huésped-Parásitos , Coccidiosis/parasitología , Coccidiosis/veterinaria , Embrión de Pollo , Transducción de Señal

RESUMEN

High-throughput tissue proteomics has great potential in the advancement of precision medicine. Here, we investigated the combined sensitivity of trap-elute microflow liquid chromatography with a ZenoTOF for DIA proteomics and phosphoproteomics. Method optimization was conducted on HEK293T cell lines to determine the optimal variable window size, MS2 accumulation time and gradient length. The ZenoTOF 7600 was then compared to the previous generation TripleTOF 6600 using eight rat organs, finding up to 23% more proteins using a fifth of the sample load and a third of the instrument time. Spectral reference libraries generated from Zeno SWATH data in FragPipe (MSFragger-DIA/DIA-NN) contained 4 times more fragment ions than the DIA-NN only library and quantified more proteins. Replicate single-shot phosphopeptide enrichments of 50-100 µg of rat tryptic peptide were analyzed by microflow HPLC using Zeno SWATH without fractionation. Using Spectronaut we quantified a shallow phosphoproteome containing 1000-3000 phosphoprecursors per organ. Promisingly, clear hierarchical clustering of organs was observed with high Pearson correlation coefficients >0.95 between replicate enrichments and median CV of 20%. The combined sensitivity of microflow HPLC with Zeno SWATH allows for the high-throughput quantitation of an extensive proteome and shallow phosphoproteome from small tissue samples.

Asunto(s)

Fosfoproteínas , Proteómica , Animales , Proteómica/métodos , Ratas , Humanos , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Células HEK293 , Fosfopéptidos/análisis , Cromatografía Líquida de Alta Presión/métodos , Proteoma/análisis , Proteoma/metabolismo

RESUMEN

Phosphorylation is the most studied post-translational modification, and has multiple biological functions. In this study, we have reanalyzed publicly available mass spectrometry proteomics data sets enriched for phosphopeptides from Asian rice (Oryza sativa). In total we identified 15,565 phosphosites on serine, threonine, and tyrosine residues on rice proteins. We identified sequence motifs for phosphosites, and link motifs to enrichment of different biological processes, indicating different downstream regulation likely caused by different kinase groups. We cross-referenced phosphosites against the rice 3,000 genomes, to identify single amino acid variations (SAAVs) within or proximal to phosphosites that could cause loss of a site in a given rice variety and clustered the data to identify groups of sites with similar patterns across rice family groups. The data has been loaded into UniProt Knowledge-Base─enabling researchers to visualize sites alongside other data on rice proteins, e.g., structural models from AlphaFold2, PeptideAtlas, and the PRIDE database─enabling visualization of source evidence, including scores and supporting mass spectra.

Asunto(s)

Genoma de Planta , Oryza , Fosfoproteínas , Proteínas de Plantas , Proteómica , Transducción de Señal , Oryza/genética , Oryza/metabolismo , Oryza/química , Proteómica/métodos , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/química , Fosfoproteínas/análisis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Fosfopéptidos/metabolismo , Fosfopéptidos/análisis , Bases de Datos de Proteínas , Secuencias de Aminoácidos , Espectrometría de Masas