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Leaf protein extraction method

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: MSV000082080 | MassIVE | Tue Feb 20 13:39:00 GMT 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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Arabidopsis Root Protein Extraction Evaluation

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

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2021-09-07 | PXD022914 | Pride

Milk bottom-up proteomics: method optimisation.

Project description:Milk is a complex fluid whose proteome displays a diverse set of proteins of high abundance such as caseins and medium to low abundance whey proteins such as ß-lactoglobulin, lactoferrin, immunoglobulins, glycoproteins, peptide hormones and enzymes. A sample preparation method that enables high reproducibility and throughput is key in reliably identifying proteins present or proteins responding to conditions such as a diet, health or genetics. Using skim milk samples from Jersey and Holstein cows, we compared three extraction procedures which have not previously been applied to samples of cows’ milk. Method A (urea) involved a simple dilution of the milk in a urea-based buffer, method B (TCA/acetone) involved a TCA/acetone precipitation and method C (methanol/chloroform) involved a tri-phasic partition method in chloroform/methanol solution. Protein assays, SDS-PAGE profiling, and trypsin digestion followed by nanoHPLC-electrospray ionisation-tandem mass spectrometry (nLC-ESI-MS/MS) analyses were performed to assess their efficiency. Replicates were used at each analytical step (extraction, digestion, injection) to assess reproducibility. Overall 186 unique accessions, major and minor proteins, were identified with a combination of methods. Method C (methanol/chloroform) yielded the best resolved SDS-patterns and highest protein recovery rates, method A (urea) yielded the greatest number of accessions, and, of the three procedures, method B (TCA/acetone) was the least compatible of all with a wide range of downstream analytical procedures. Our results also highlighted breed differences between the proteins in milk of Jersey and Holstein cows.

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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

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UPA-Seq: Prediction of functional lncRNAs using the sensitivities to UV-crosslinking

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Genomewide demarcation of RNA PolII transcription units by physical fractionation of chromatin- Neutral enrichment

Project description:Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA. We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III. Noncoding regions could be further fractionated into promoters and segments lacking promoters. The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions. The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes. Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states. Keywords: Genomewide mapping of regulatory elements through differential fractionation of crosslinked chromatin based on nucleosome occupancy. Neutral enrichment- All DNA was prepared by glass-bead disruption and standard phenol-chloroform extraction with the modification that the cells were first broken in the absence of phenol-chloroform. The extract was centrifuged for 5 s at 14,000Xg, and the supernatant was sonicated and subsequently phenol-chloroform extracted.

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An optimised protocol for protein extraction from salt gland-enriched tissues of a mangrove tree species for proteomic analysis

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