Electroblotting through a tryptic membrane for LC-MS/MS analysis of proteins separated in electrophoretic gels
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Escherichia Coli (ncbitaxon:562)
SUBMITTER: 
Adrianna Bickner
PROVIDER: MSV000086010 | MassIVE | Tue Aug 25 09:13:00 BST 2020
REPOSITORIES: MassIVE
Similar Datasets
Project description:Digestion of proteins separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) remains a popular method for protein identification using mass-spectrometry based proteomics. Although robust and routine, the in-gel digestion procedure is laborious and time-consuming. Electroblotting to a capture membrane prior to digestion reduces preparation steps but requires on-membrane digestion that yields fewer peptides than in-gel digestion. This paper develops direct electroblotting through a trypsin-containing membrane to a capture membrane to simplify extraction and digestion of proteins separated by SDS-PAGE. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies the extracted peptides. Analysis of peptides from different capture membrane pieces shows that electrodigestion does not greatly disturb the spatial resolution of a standard protein mixture separated by SDS-PAGE. Electrodigestion of an Escherichia coli (E. coli) cell lysate requires four hours of total sample preparation and results in only 13% fewer protein identifications than in-gel digestion, which can take 24 h. Compared to simple electroblotting and protein digestion on a poly(vinylidene difluoride) (PVDF) capture membrane, adding a trypsin membrane to the electroblot increases the number of protein identifications by 22%. Additionally, electrodigestion experiments using capture membranes coated with polyelectrolyte layers identify a higher fraction of small proteolytic peptides than capture on PVDF or in-gel digestion.
| S-EPMC7704035 | biostudies-literature
Project description:Proteins from E. coli outer membrane were extracted with carbonate method, separated in 2D-PAGE and transferred onto nitrocellulose membrane. After incubation with flourescent-labelled mucin, mucin-binding protein spots were cut out, subjected to trypsinolysis and identified by protein analysis with HPLC-MS-MS.
2019-12-16 | PXD010472 | Pride
Project description:PURPOSE:Early diagnosis of primary immunodeficiency disorders (PIDDs) is critical for maximizing patient survival and clinical outcomes. Consequently, there is significant interest in developing broad-based, high-throughput, screening approaches capable of utilizing small blood volumes to identify patients with PIDD. EXPERIMENTAL DESIGN:We developed a novel proteomic screening approach using tandem mass spectrometry to simultaneously identify specific signature peptides derived from the transmembrane protein cluster of differentiation 3 (CD3)? and the intracellular proteins Wiskott-Aldrich syndrome protein (WASP) and Bruton's tyrosine kinase (BTK) as markers of three life-threatening PIDDs; severe combined immunodeficiency, Wiskott-Aldrich syndrome, and X-linked Agammaglobulinemia. Signature peptides were analyzed by LC/MS-MS in proteolytically digested lysates from cell lines and white blood cells (WBCs). The amount of each peptide was determined by the ratio of the signature peptide peak area to that of a known amount of labeled standard peptide. Peptide concentrations were normalized to actin. RESULTS:We show that signature peptides from CD3?, WASP, and BTK were readily detected in proteolytically digested cell lysate and their absence could correctly identify PIDD patients. CONCLUSIONS AND CLINICAL RELEVANCE:This proof of concept study demonstrates the applicability of this approach to screen for PIDD and raises the possibility that it could be further multiplexed to identify additional PIDDs and potentially other disorders.
| S-EPMC3489469 | biostudies-literature
Project description:In addition to nucleic acids, a variety of biomolecules have been found on the plasma membrane. Whereas, researchers have realized that RNA has the ability to bind to membrane vesicles in vitro and connect to the plasma membrane of the cell. Hence, the combination of high-throughput sequencing and in suit labelling methods provides approaches for large-scale identification of subcellular RNAs. Here, we applied the recently published method APEX-seq, and identified 75 plasma membrane-associated RNAs, of which lncRNA PMAR72 is located around the membrane. In addition, we observed that PMAR72 was concentrated into foci in specific membrane areas. Our findings will provide some new evidence to elaborate the relationship between RNA and the plasma membrane of mammalian cells.
2021-05-25 | GSE166725 | GEO
Project description:Classically, intracellular nuclear androgen receptors (ar) are considered to be the key mediators of androgen actions. However, there have been several reports that androgens can also exert their effects by binding to integral membrane proteins. Despite in vitro cloning and characterization of several putative membrane androgen receptors, their functions in vivo remain poorly understood. We used a chemical-genetic screen in zebrafish and discovered that the G-protein coupled receptor, GPRC6A, mediates androgen action during embryonic development. We exposed zebrafish embryos to testosterone (30 μM) from 2-4 hours post-fertilization (hpf) until 72 hpf. We found that testosterone exposure caused cardiac edema in wild-type and ar mutant zebrafish embryos, but there was a significant reduction in this phenotype in the grprc6a mutant embryos, suggesting that gprc6a regulates androgen action independently of nuclear androgen receptors. Additionally, we exposed wild-type embryos to testosterone together with GPRC6A antagonists and observed a significant suppression of the cardiac edema phenotype. These results suggest that testosterone causes cardiac edema in zebrafish embryos by acting via the integral membrane protein GPRC6A, independently of nuclear androgen receptors. Overall, our study provides insights into non-genomic androgen signaling during embryonic development and identifies GPRC6A as a key receptor mediating androgen action.
2024-09-12 | GSE274852 | GEO
Project description:Protein band obtained from the Panax notoginseng leaves homogenate following an activity-guided protein-purification procedure, that showed regioselective ginsenoside hydrolysis activity was digested with trypsin and analyzed by Nano LC-LTQ Orbitrap MS to construct a peptide library of accessible amino acid sequences. Subsequent mutual sequence alignment analysis between the peptide and transcript libraries identified three candidate genes.
2024-01-05 | PXD048221 | Pride
Project description:There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-?-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90?min. In a blind set, the assay detected 21/24 bla NDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (?0.1 fm/?g) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the bla NDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.
| S-EPMC6709477 | biostudies-literature
Project description:In recent decades, proteinic drugs, in particular monoclonal antibodies, are taking the leading role of small molecule drugs, and peptide mapping relying on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach to substitute the role of a ligand-binding assay for the quality control of the proteinic drugs. However, such LC-MS/MS approaches extensively suffer from time-intensive measurements, leading to a limited throughput. To achieve accelerated measurements, here, the potential of DI-MS/MSALL towards tryptic peptide mapping was evaluated through comparing with well-defined LC-MS/MS means, and human serum albumin (HSA) was employed as the representative protein for applicability illustration. Among the 55 tryptic peptides theoretically suggested by Skyline software, 47 were successfully captured by DI-MS/MSALL through acquiring the desired MS2 spectra, in comparison to 51 detected by LC-MS/MS. DI-MS/MSALL measurements merely took 5 min, which was dramatically superior to the LC-MS/MS assay. Noteworthily, different from fruitful multi-charged MS1 signals for LC-MS/MS, most quasi-molecular ions received lower charged states. DI-MS/MSALL also possessed advantages such as lower solvent consumption and facile instrumentation; however, more sample was consumed. In conclusion, DI-MS/MSALL is eligible to act as an alternative analytical tool for LC-MS/MS towards the peptide mapping of proteinic drugs, particularly when a heavy measurement workload.
| S-EPMC8963265 | biostudies-literature
Project description:The Bin/amphiphysin/Rvs (BAR) domain protein FAM92A1 is a multifunctional protein engaged in the regulation of mitochondrial ultrastructure and ciliogenesis. However, the physiological role of FAM92A1 in the brain, particularly in neurons with specialized membrane architecture, remains unexplored. Here, we demonstrate that FAM92A1 is prominently expressed in the brain and neurons during early mouse embryonic development. FAM92A1 knockout mice display brain morphology alterations, age-associated memory decline, and cognitive deficits. Moreover, the absence of FAM92A1 triggers hippocampal neuron degeneration and disrupts neuronal complexity and synaptic plasticity. Crucially, FAM92A1 deficiency leads to anomalies in membrane remodeling and disturbances in neuronal endocytic processes. Through analysis of the crystal structure of the FAM92A1 BAR domain, we unveil its intrinsic capability to form dimers, contributing to a distinctive curved conformation critical for membrane remodeling. By combining structural and molecular simulation, we further gain a comprehensive understanding of how FAM92A1 interacts with membranes to induce lipid clustering and generate membrane curvature. In summary, our study provides valuable insights into the physiological function of FAM92A1 in the brain. It uncovers the molecular mechanisms through which FAM92A1 regulates synaptic plasticity and neural function by influencing membrane remodeling and endocytic processes.
2024-07-05 | GSE264202 | GEO
Project description:Mouse hippocampus membrane fractions were prepared using sucrose density gradient ultracentrifugation. Membrane proteins were solubilized using three different condition; 6-ACA/Triton X-100 or ProteoExtract Native Membrane Protein Extraction Kit (Calbiochem, Cat. No. 444810) or ProteoExtract Transmembrane Protein Extraction Kit (Novagen, Cat. No. 71772-3). Solubilized membrane proteins were separated on Blue Native PAGE or SDS-PAGE. Protein bands were excised, destained for peptide sample preparation. Proteins were reduced (DTT), alkylated (iodoacetamide), and in-gel digested with chymotrypsin or trypsin. Digested peptides were extracted then subjected to nanoHPLC and tandem MS analysis with Thermo Orbitrap Velos Pro mass spectrometer.
2014-08-15 | PXD000311 | Pride