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Arabidopsis Root Protein Extraction Evaluation

ABSTRACT: We evaluated several approaches for sample processing that are based on popular urea extraction, methanol/chloroform, and phenol methods. Additionally, we examined whether these methods were improved using FASP-based on filter digestion. We found that phenol-FASP and UA-FASP methods enabled the best coverage of protein, peptides, and MS/MS.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702)

SUBMITTER: Justin Walley

PROVIDER: MSV000082362 | MassIVE | Tue May 15 12:30:00 BST 2018

REPOSITORIES: MassIVE

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Publications

Assessment and Refinement of Sample Preparation Methods for Deep and Quantitative Plant Proteome Profiling.

Song Gaoyuan G Hsu Polly Yingshan PY Walley Justin W JW

Proteomics 20180827 17

A major challenge in the field of proteomics is obtaining high-quality peptides for comprehensive proteome profiling by LC-MS. Here, evaluation and modification of a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissue are done. It was found that inclusion of filter-aided sample preparation (FASP) based on filter digestion improves all protein extraction methods tested. Ultimately, a detergent-free urea-FASP approach that enables deep and robust quantificat ...[more]

PMID: 30035338

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Leaf protein extraction method

Project description:We evaluated several approaches for sample processing that are based on popular urea extraction, methanol/chloroform, and phenol methods. Additionally, we examined whether these methods were improved using FASP-based on filter digestion. We found that phenol-FASP and UA-FASP methods enabled the best coverage of protein, peptides, and MS/MS

2018-02-20 | MSV000082080 | MassIVE

Parallel protein extraction methods increase the diversity of protein identification in Park Grass Experiment soil

Project description:We have combined a modified protein extraction method, heat/thaw/phenol/chloroform (HTPC), with the established Surfactant extraction method to identify proteins from Park Grass Experiment (PGE) soil, which has an extensively sequenced microbial database.

2022-09-20 | PXD017392 | Pride

Optimization of Protein Isolation and Label–free Quantitative Proteomic Analysis in Four Different Tissues of Korean Ginseng

Project description:Korean ginseng is one of the most valuable medicinal plants worldwide. Yet, our understanding of ginseng proteomics is largely limited due to difficulties in extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone–MeOH/chloroform, Phenol–TCA/acetone, and Phenol–MeOH/chloroform methods. Consequently, the TCA/acetone–MeOH/chloroform method displayed the highest extraction efficiency, thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label–free quantitative proteomics approach. This approach led to the identification of 2,604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis including the methylerythritol 4–phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP–glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ–based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.

2021-09-07 | PXD022914 | Pride

Evaluation of Two Simultaneous Metabolomic and Proteomic Extraction Protocols Assessed by Ultra-High-Performance Liq-uid Chromatography Tandem Mass Spectrometry

Project description:Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis and for a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and pro-teomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently used solvent systems: chloro-form/methanol and methanol-only. Whole blood samples were collected from participants (n=6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods i. methanol precipitation and, ii. 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.

2023-01-17 | PXD038750 | Pride

Evaluation of Protein Extraction Methods to Improve Meta-Proteomics Analysis of Treated Wastewater Biofilms

Project description:Metaproteomics can be used to study functionally active biofilm-based bacterial populations in reclaimed water distribution systems, which in turn result in bacterial regrowth that impacts the water quality. However, existing protein extraction methods have differences in their protein recovery and have not been evaluated for their efficacies in reclaimed water biofilm samples. In this study, we first evaluated six different protein extraction methods with diverse chemical and physical properties on a mixture of bacterial cell culture. Based on a weighting scores-based evaluation, the extraction protocols in order of decreasing performance are listed as B-PER > RIPA > PreOmics > SDS > AllPrep > Urea. The highest four optimal methods on cell culture were further tested against treated wastewater non-chlorinated and chlorinated effluent biofilms. In terms of protein yield, our findings showed that RIPA performed the best; however, the highest number of proteins were extracted from SDS and PreOmics. Furthermore, SDS and PreOmics worked best to rupture gram-positive and gram-negative bacterial cell walls. Considering the five evaluation factors, PreOmics obtained highest weighted score, indicating its potential effectiveness in extracting proteins from biofilms. This study provides the first insight into evaluating protein extraction methods to facilitate metaproteomics for complex reclaimed water matrices.

2023-08-04 | PXD042292 | Pride

Project description:Bacterial diveristy of Sardinella gut microbiome: Phenol:chloroform method

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Three protein extraction methods for mass spectrometry proteomics of fresh frozen mouse tissue

Project description:We present three different methods for protein extraction from fresh frozen mouse heart tissue: 1) extraction using SDS buffer followed by FASP purification, 2) extraction using SDS buffer followed by S-Trap purification, and 3) extraction using a guanidine hydrochloride buffer followed by in-solution digestion. Based on bicinchoninic acid assay, all three methods display similar recovery of total protein content. However, proteomics-based analysis identified far fewer proteins from the extraction guanidine hydrochloride. SDS-FASP identifies as many proteins as the SDS-STrap method with good coverage of proteins from different cellular compartments.

2020-07-30 | PXD020256 | Pride

Evaluation of methods for modeling transcription factor sequence specificity

Project description:Genomic analyses often involve scanning for potential transcription-factor (TF) binding sites using models of the sequence specificity of DNA binding proteins. Many approaches have been developed to model and learn a protein’s binding specificity by representing sequence motifs, including the gaps and dependencies between binding-site residues, but these methods have not been systematically compared. Here we applied 26 such approaches to in vitro protein binding microarray data for 66 mouse TFs belonging to various families. For 9 TFs, we also scored the resulting motif models on in vivo data, and found that the best in vitro–derived motifs performed similarly to motifs derived from in vivo data. Our results indicate that simple models based on mononucleotide position weight matrices learned by the best methods perform similarly to more complex models for most TFs examined, but fall short in specific cases. In addition, the best-performing motifs typically have relatively low information content, consistent with widespread degeneracy in eukaryotic TF sequence preferences.

2012-12-13 | GSE42864 | GEO

Evaluation of methods for modeling transcription factor sequence specificity

2012-12-13 | E-GEOD-42864 | biostudies-arrayexpress

Evaluation of Two Simultaneous Metabolomic and Proteomic Extraction Protocols Assessed by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry

Project description:Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis and for a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and proteomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently used solvent systems: chloro-form/methanol and methanol-only. Whole blood samples were collected from participants (n=6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods i. methanol precipitation and, ii. 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.

2022-12-06 | ST002391 | MetabolomicsWorkbench

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