Proteins and proteomes are dynamic and complex. The accurate identification and measurement of their properties such as abundance, location, and turnover are challenging tasks. Even though ...high-throughput proteomics has significantly evolved, the technique still lacks fully quantitative and reproducible qualities. A mass spectrometry-based targeted proteomic strategy called selective reaction monitoring (SRM) has emerged in recent years as an important multiplex platform to precisely quantify sets of proteins in multiple samples. This has several advantages in terms of sensitivity, reproducibility, and sample consumption compared to classical methods including those based on antibodies. Here, we present a detailed protocol for quantitation of panels of proteins from cell line extracts using the SRM targeted proteomics approach.
Current proteomics technologies allow substantial depth of analysis of cellular and subcellular proteomes as shown in the proteomic profiling of ovarian cancer cells. This in-depth analysis has ...elucidated the repertoire of proteins expressed on the cell surface and proteins released into the extracellular milieu, uncovering extensive shedding of extracellular domains of cell adhesion proteins and a highly dynamic protein secretion process. The protein sets identified provide a rich resource of potential circulating markers and targets for imaging and therapeutics for ovarian cancer.
Contrary to what is being said by several colleagues (and even advertised), data‐independent analysis/acquisition (DIA) is not a new mass spectrometry acquisition method. Here we draw a timeline of ...events showing that DIA has been around since the early 2000s.
The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome ...inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.
Phosphorylation is one of the most dynamic and widespread post‐translational modifications regulating virtually every aspect of eukaryotic cell biology. Here, we assemble a dataset from 75 ...independent phosphoproteomic experiments performed in our laboratory using Saccharomyces cerevisiae. We report 30,902 phosphosites identified from cells cultured in a range of DNA damage conditions and/or arrested in distinct cell cycle stages. To generate a comprehensive resource for the budding yeast community, we aggregate our dataset with the Saccharomyces Genome Database and another recently published study, resulting in over 46,000 budding yeast phosphosites. With the goal of enhancing the identification of functional phosphorylation events, we perform computational positioning of phosphorylation sites on available 3D protein structures and systematically identify events predicted to regulate protein complex architecture. Results reveal hundreds of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic “clashes” predicted to disrupt the interaction. With the advancement of Cryo‐EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.
SYNOPSIS
This study compiles a large set of independent experiments into a comprehensive phosphoproteome resource for the budding yeast community. 3D analysis of protein interaction interfaces and other strategies are used to predict functionality amongst the ≥ 40,000 reported phosphorylation events.
75 independent phosphoproteomic experiments were consolidated into a comprehensive resource of over 40,000 budding yeast phosphorylation sites.
Multiple strategies were used to infer functional phosphorylation events.
Mapping phosphorylation sites to protein interaction interfaces revealed phosphorylation sites that regulate protein‐protein interactions.
This study compiles 75 independent SILAC‐based experiments into a comprehensive phosphoproteome resource for budding yeast. 3D analysis of protein interaction interfaces and other strategies are used to predict functionality amongst the ≥ 40,000 reported phosphorylation events.
We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung ...cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models.
► Organ-type specific protein signatures found in plasmas of mouse models of cancer ► Plasma protein signatures reflect molecularly distinct subtypes of lung cancer ► Networks of plasma proteins inform about genes that drive tumor development ► Concordant findings in human lung cancer blood samples based on ELISAs
Patients with psychiatric disorders exhibit dysfunctions in peripheral and central metabolism. This may be a root cause of impaired neuronal function, manifested as changes in mood, behavior, and ...cognitive capabilities in patients suffering with these conditions. Here we describe a selective reaction monitoring mass spectrometry (SRM-MS)-based targeted proteomic protocol for precise simultaneous quantitation of three glycolytic enzymes in postmortem brain tissue extracts. The SRM-MS approach has several advantages in terms of sensitivity, reproducibility, and reduced sample consumption, compared to traditional MS methods.
Systematic searches for plasma proteins that are biological indicators, or biomarkers, for cancer are underway. The difficulties caused by the complexity of biological-fluid proteomes and tissue ...proteomes (which contribute proteins to plasma) and by the extensive heterogeneity among diseases, subjects and levels of sample procurement are gradually being overcome. This is being achieved through rigorous experimental design and in-depth quantitative studies. The expected outcome is the development of panels of biomarkers that will allow early detection of cancer and prediction of the probable response to therapy. Achieving these objectives requires high-quality specimens with well-matched controls, reagent resources, and an efficient process to confirm discoveries through independent validation studies.
Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ...ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases in the canonical ubiquitination pathway, here we show that 2 paralogous Legionella effectors, Lpg2154 (DupA; deubiquitinase for PRubiquitination) and Lpg2509 (DupB), reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these 2 genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella-containing vacuoles. Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.
The outbreak of the COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome of Coronavirus 2 (SARS-CoV-2), has fueled the search for diagnostic tests aiming at the control and reduction of the ...viral transmission. The main technique used for diagnosing the Coronavirus disease (COVID-19) is the reverse transcription-polymerase chain reaction (RT-PCR) technique. However, considering the high number of cases and the underlying limitations of the RT-PCR technique, especially with regard to accessibility and cost of the test, one does not need to overemphasize the need to develop new and less expensive testing techniques that can aid the early diagnosis of the disease. With that in mind, we developed an ultrasensitive magneto-assay using magnetic beads and gold nanoparticles conjugated to human angiotensin-converting enzyme 2 (ACE2) peptide (Gln24-Gln42) for the capturing and detection of SARS-CoV-2 Spike protein in human saliva. The technique applied involved the use of a disposable electrochemical device containing eight screen-printed carbon electrodes which allow the simultaneous analysis of eight samples. The magneto-assay exhibited an ultralow limit of detection of 0.35 ag mL−1 for the detection of SARS-CoV-2 Spike protein in saliva. The magneto-assay was tested in saliva samples from healthy and SARS-CoV-2-infected individuals. In terms of efficiency, the proposed technique – which presented a sensitivity of 100.0% and specificity of 93.7% for SARS-CoV-2 Spike protein-exhibited great similarity with the RT-PCR technique. The results obtained point to the application potential of this simple, low-cost magneto-assay for saliva-based point-of-care COVID-19 diagnosis.
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•Ultrasensitive magneto-assay for SARS-CoV-2 Spike detection in saliva•Disposable screen-printed carbon electrode array for analysis of multiples samples•The Spike detection in saliva samples showed good correlation with RT-PCR technique.•The magneto-assay showed excellent clinical sensibility and specificity for COVID-19 diagnosis
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