LC-MS/MS profiling of sample processing optimization of formalin-fixed, paraffin embedded tissues
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Breast Cancer Cell
DISEASE(S): Breast Cancer
SUBMITTER: 
Jacob Kennedy
LAB HEAD: Paulovich, Amanda G
PROVIDER: PXD003770 | Pride | 2016-07-25
REPOSITORIES: Pride
Similar Datasets
Project description:The proteomics of archival samples has advanced significantly during the last two decades, making possible the use of vast FFPE tissue archives in biomarker studies. However, due to the limited data regarding comparison of the performance of different protocols, there is currently no consensus on the most effective protocol for shotgun proteomic analysis of FFPE tissue. In-solution digestion method using Rapigest as detergent and FASP method, both reported to deliver superior results on FFPE tissues were compared using two label-free data-independent LC-MS/MS acquisition modes. Archival FFPE prostate tissues stored 7 years and mirroring fresh frozen samples were processed with both protocols in 3 technical replicates. Data were comparatively evaluated in terms of protein and peptide identifications in fresh frozen and FFPE samples, analysis of the fresh and FFPE tissues representative proteome (molecular weight distribution, distribution according to protein’s pI, cellular component analysis of proteins and quantitation accuracy) and technical reproducibility of the Rapigest and FASP protocols.
2021-01-15 | PXD021074 | Pride
Project description:The DNA-damage response (DDR) is a critical network for maintaining genomic integrity, and mutations in the DDR are among the most frequently identified in tumors. Phosphorylation is a key signaling event in the DDR network. We sought to design high throughput, mass spectrometry based assays to monitor phosphopeptides in the DDR network for biological experiments and pharmacodynamics studies. These assays require an enrichment step, which can be done using antibodies. Developing antibodies de novo for phosphopeptide enrichment is costly and time-consuming, so we assessed the feasibility of immobilized-metal affinity chromatography (IMAC) enrichment coupled with multiple reaction monitoring-mass spectrometry (MRM-MS) for precise measurement of large numbers of phosphopeptides in the DDR network. Towards this goal, we evaluate the ability of the approach to reproducibly measure the endogenous phosphorylation levels of proteins associated with the DDR. Reproducibly detectable phosphopeptide targets for MRM-based assay development were empirically identified from LC-MS/MS analyses of SILAC-labeled MCF10A human breast epithelial cells exposed or mock-exposed to DNA damage. Samples were treated in triplicate with either ionizing radiation (IR) or methyl methanesulfonate (MMS) as the DNA-damaging agent. SILAC-labeled cells were prepared in two separate (label swap) experiments. First, treated cell lines were grown in heavy SILAC medium and untreated in light, and second, this labeling scheme was reversed. Untreated and treated cells were mixed at a 1:1 ratio by protein mass. To identify phosphopeptides at levels observable by MRM in an approach amenable to moderate throughput, a single step IMAC enrichment was developed for rapid processing of whole cell lysate.
2015-12-01 | PXD002363 | Pride
Project description:Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria, the results indicate a success rate up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents.
2016-04-13 | PXD003370 | Pride
Project description:Background: The horn fly, Haematobia irritans (L.), is an obligate blood-feeding parasite of cattle and control of this pest is a continuing problem in the United States and other parts of the world. Worldwide annual economic losses attributable to this pest surpass $1 billion. The fly is becoming resistant to pesticides and new control technologies are needed by cattle producers. Results: Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene. To identify candidate genes to supply this gene promoter, microarrays were designed from a recently developed horn fly EST database and probed to identify female-specific and larval-specific differential gene expression. Analysis of dyeswap experiments found 432 and 417 transcripts which were over- and under-expressed in adult female flies, respectively, compared to adult male flies. Additionally, 419 and 871 transcripts were over- and under-expressed in first instar larvae compared to adult flies. Three transcripts were identified which were over-expressed in adult females flies compared to adult males and which also were over-expressed in the first instar larval lifestage compared to adult flies. Conclusion: We have identified 3 strong candidates for further evaluation as a gene promoter source for the development of a female-specific conditional lethality system in the horn fly. One of these candidates, the putative nanos orthologue, has a high female-to-male expression ratio, has a moderate expression level in first instar larvae, and has been well characterized in D. melanogaster. Further investigations leading from this microarray analysis will include transformation of the horn fly and evaluation of the female conditional lethal system components for applicability to the specific biological parameters of natural populations of the horn fly. Keywords: evaluation of 1st instar larvae genes vs pooled adult + egg genes In short, RNA from female horn flies was labeled and hybridized against labeled RNA from male horn flies in a dyeswap design. Similarly, RNA from first instar larvae was labeled and hybridized against labeled a 50:50 mix of RNA from adult male and female flies. All labeling, hybridization and washing procedures were performed according to respective manufacturer protocols. For each sample, 10 ïg of total RNA and 50-fold dilution of Agilent two color spike-in control RNA kit (Agilent Technologies Inc.) were labeled with either CyDye3-dCTP or CyDye5-dCTP (Amersham Biosciences, Piscataway, NJ) using the LabelStar kit (Qiagen Inc., Valencia, CA) and oligo-dT and Random nonamers (Sigma-Aldrich Inc., St. Louis, MO). Labeled cDNA was hybridized to the Agilent microarrays using the Gene Expression Hybridization kit (Agilent Technologies Inc.) following manufacturerâs protocols. Arrays were washed with Gene Expression Wash Buffer kit (Agilent Technologies Inc.) A total of 8 arrays, including a dye swap for each RNA replicate (4 biological replicates), were analyzed to obtain genes that were consistently regulated while limiting false discover rate (FDR) below 2.5% [26].
2010-05-26 | E-GEOD-11364 | biostudies-arrayexpress
Project description:Background: The horn fly, Haematobia irritans (L.), is an obligate blood-feeding parasite of cattle and control of this pest is a continuing problem in the United States and other parts of the world. Worldwide annual economic losses attributable to this pest surpass $1 billion. The fly is becoming resistant to pesticides and new control technologies are needed by cattle producers. Results: Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene. To identify candidate genes to supply this gene promoter, microarrays were designed from a recently developed horn fly EST database and probed to identify female-specific and larval-specific differential gene expression. Analysis of dyeswap experiments found 432 and 417 transcripts which were over- and under-expressed in adult female flies, respectively, compared to adult male flies. Additionally, 419 and 871 transcripts were over- and under-expressed in first instar larvae compared to adult flies. Three transcripts were identified which were over-expressed in adult females flies compared to adult males and which also were over-expressed in the first instar larval lifestage compared to adult flies. Conclusion: We have identified 3 strong candidates for further evaluation as a gene promoter source for the development of a female-specific conditional lethality system in the horn fly. One of these candidates, the putative nanos orthologue, has a high female-to-male expression ratio, has a moderate expression level in first instar larvae, and has been well characterized in D. melanogaster. Further investigations leading from this microarray analysis will include transformation of the horn fly and evaluation of the female conditional lethal system components for applicability to the specific biological parameters of natural populations of the horn fly. Keywords: adult male vs female hornfly gene expression In short, RNA from female horn flies was labeled and hybridized against labeled RNA from male horn flies in a dyeswap design. Similarly, RNA from first instar larvae was labeled and hybridized against labeled a 50:50 mix of RNA from adult male and female flies. All labeling, hybridization and washing procedures were performed according to respective manufacturer protocols. For each sample, 10 ïg of total RNA and 50-fold dilution of Agilent two color spike-in control RNA kit (Agilent Technologies Inc.) were labeled with either CyDye3-dCTP or CyDye5-dCTP (Amersham Biosciences, Piscataway, NJ) using the LabelStar kit (Qiagen Inc., Valencia, CA) and oligo-dT and Random nonamers (Sigma-Aldrich Inc., St. Louis, MO). Labeled cDNA was hybridized to the Agilent microarrays using the Gene Expression Hybridization kit (Agilent Technologies Inc.) following manufacturerâs protocols. Arrays were washed with Gene Expression Wash Buffer kit (Agilent Technologies Inc.) A total of 8 arrays, including a dye swap for each RNA replicate (4 biological replicates), were analyzed to obtain genes that were consistently regulated while limiting false discover rate (FDR) below 2.5% [26].
2010-05-26 | E-GEOD-11363 | biostudies-arrayexpress
Project description:Proteomics is a high-throughput technology which has been developed with the aim of investigating the maximum number of proteins in cells in a given experiment. However, protein discovery and data generation vary in depth and coverage when different technical strategies are selected. In this study, three different sample preparation approaches, and peptide or protein fractionation methods, were applied to identify and quantify proteins from log-phase yeast lysate: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), filter-aided sample preparation coupled with gas phase fractionation (FASP-GPF), and FASP - high pH reversed phase fractionation (HpH). Fractions were initially analyzed and compared using nanoflow liquid chromatography – tandem mass spectrometry (nanoLC-MS/MS) employing data dependent acquisition on a linear ion trap instrument. The number of fractions and analytical replicates was adjusted so that each experiment used a similar amount of mass spectrometric instrument time. A second set of experiments was performed using a Q Exactive Orbitrap instrument, comparing FASP-GPF, SDS-PAGE and FASP-HpH. Compared with results from the linear ion trap mass spectrometer, the use of a Q Exactive Orbitrap mass spectrometer enabled a small increase in protein identifications, and a very large increase in peptide identifications. This shows that the main advantage of using the higher resolution instrument is in increased proteome coverage. A total of 1035, 1357 and 2134 proteins were separately identified by FASP-GPF, SDS-PAGE, and FASP-HpH. Combining results from the Orbitrap experiments, there were a total of 2269 proteins found, with 94% of them identified using the FASP-HpH method. Therefore, the FASP-HpH method is the optimal choice among these approaches, when applied to this type of sample.
2020-11-30 | PXD018854 | Pride
Project description:Existing protocols for full-length single-cell RNA sequencing (scRNA-seq) produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single nucleotide polymorphisms, and allow assembly of the VDJ region of the T- and B-cell receptor sequences. Since full length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development for instance. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared to droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length scRNA-seq, including both an in-house automated SMART-seq2 protocol, and a commercial kit-based workflow. We discuss these two protocols in terms of ease-of-use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we propose an optimized in-house automated protocol with dramatically reduced cost. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative (www.humancellatlas.org) and are available on protocols.io.
2020-12-31 | E-MTAB-9345 | biostudies-arrayexpress
Project description:In this experiment, we've examined chromatin conformation of OG2 (B6; CBA-Tg(Pou5f1-EGFP)2Mnn/J; stock number 004654) mouse stem cells cultured as described in (Shi et al., 2008), using different amounts of starting cells. We performed a modified in situ Hi-C protocol for 6 samples digested with MboI restriction enzyme having as starting material 1 million (M), 100 thousand (k), 50k, 25k, 10k or 1k cells. As well as, to 2 samples digested with HindIII restriction enzyme that had as starting material 5M or 100k cells. Traditional in situ Hi-C protocols recommend 5-10 million starting cells. The aim of the experiment was to assess the impact of decreasing the cell number on reproducibility, library complexity, chromatin structure visualization in order to adapt the method to the study of rare cell populations. Furthermore, we have characterised the 3D structure of peripheral blood mononuclear cells (PBMCs) obtained from a blood extraction from a healthy donor and from a lymph node biopsy from a DLBCL patient as a proof of concept for the suitability of Low-C for rare cell population analysis.
2018-11-30 | E-MTAB-5875 | biostudies-arrayexpress
Project description:The aim of the MS analysis is to identify fatty acids modifying Cys3 of GNAI3. Detailed description in the manuscript Nuskova et al. submitted to NatComms.
2021-07-22 | PXD021517 | Pride
Project description:The aim of the MS analysis is to identify fatty acids modifying Cys3 of GNAI3 in cells exposed to high C16:0 or C18:0 concentrations. Detailed description in the manuscript Nuskova et al. submitted to NatComms.
2021-08-26 | PXD027064 | Pride