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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

SUBMITTER: Justin Walley

PROVIDER: MSV000082080 | MassIVE | Tue Feb 20 13:39:00 GMT 2018

REPOSITORIES: MassIVE

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			Action	DRS
	MSV000082080	Other
	params.xml	Xml
	statistics.tsv	Tabular
	GS-110717-PhenolFASP-39-LF-Trp-1ug-1D-QE2.raw	Raw
	GS-110717-UAFASP-38-LF-Trp-1ug-1D-QE2.raw	Raw

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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

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.

2018-05-15 | MSV000082362 | MassIVE

Telomere length varies by DNA extraction method: implications for epidemiologic research.

Project description:Both shorter and longer telomeres in peripheral blood leukocyte (PBL) DNA have been associated with cancer risk. However, associations remain inconsistent across studies of the same cancer type. This study compares DNA preparation methods to determine telomere length from patients with colorectal cancer.We examined PBL relative telomere length (RTL) measured by quantitative PCR (qPCR) in 1,033 patients with colorectal cancer and 2,952 healthy controls. DNA was extracted with phenol/chloroform, PureGene, or QIAamp.We observed differences in RTL depending on DNA extraction method (P < 0.001). Phenol/chloroform-extracted DNA had a mean RTL (T/S ratio) of 0.78 (range 0.01-6.54) compared with PureGene-extracted DNA (mean RTL of 0.75; range 0.00-12.33). DNA extracted by QIAamp yielded a mean RTL of 0.38 (range 0.02-3.69). We subsequently compared RTL measured by qPCR from an independent set of 20 colorectal cancer cases and 24 normal controls in PBL DNA extracted by each of the three extraction methods. The range of RTL measured by qPCR from QIAamp-extracted DNA (0.17-0.58) was less than from either PureGene or phenol/chloroform (ranges, 0.04-2.67 and 0.32-2.81, respectively).RTL measured by qPCR from QIAamp-extracted DNA was less than from either PureGene or phenol/chloroform (P < 0.001).Differences in DNA extraction method may contribute to the discrepancies between studies seeking to find an association between the risk of cancer or other diseases and RTL.

2013-01-01 | S-EPMC3827976 | BioStudies

Low-cost RNA extraction method for highly scalable transcriptome studies.

Project description:RNA extraction has been improved by integration of a variety of materials in the protocol, such as phenol, guanidine thiocyanate, and silica, according to the case-specific demands. However, few methods have been designed for high-throughput RNA preparation for large-scale transcriptome studies. In this study, we established a high-throughput guanidinium thiocyanate and isopropyl alcohol based RNA extraction method (HighGI). HighGI is based on simple and phenol-free homemade buffers and the cost is substantially lower than a column-based commercial kit. We demonstrated that the quality and quantity of RNA extracted with HighGI were comparable to those extracted with a conventional phenol/chloroform-based method and a column-based commercial kit. HighGI retained small RNAs less than 200 bp, which are lost with a commercial column-based kit. We also demonstrated that HighGI is readily applicable to semi-automated RNA extraction. HighGI enables high-throughput RNA extraction for large-scale RNA preparation with high yield and quality.

2020-01-01 | S-EPMC7495201 | BioStudies

An Efficient Single Phase Method for the Extraction of Plasma Lipids.

Project description:Lipidomic approaches are now widely used to investigate the relationship between lipid metabolism, health and disease. Large-scale lipidomics studies typically aim to quantify hundreds to thousands of lipid molecular species in a large number of samples. Consequently, high throughput methodology that can efficiently extract a wide range of lipids from biological samples is required. Current methods often rely on extraction in chloroform:methanol with or without two phase partitioning or other solvents, which are often incompatible with liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS). Here, we present a fast, simple extraction method that is suitable for high throughput LC ESI-MS/MS. Plasma (10 ?L) was mixed with 100 ?L 1-butanol:methanol (1:1 v/v) containing internal standards resulting in efficient extraction of all major lipid classes (including sterols, glycerolipids, glycerophospholipids and sphingolipids). Lipids were quantified using positive-ion mode LC ESI-MS/MS. The method showed high recovery (>90%) and reproducibility (%CV < 20%). It showed a strong correlation of all lipid measures with an established chloroform:methanol extraction method (R2 = 0.976). This method uses non-halogenated solvents, requires no drying or reconstitution steps and is suitable for large-scale LC ESI-MS/MS-based lipidomic analyses in research and clinical laboratories.

1000-01-01 | S-EPMC4495379 | BioStudies

High abundance of ammonia-oxidizing Archaea in coastal waters, determined using a modified DNA extraction method.

Project description:Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.

1000-01-01 | S-EPMC2849251 | BioStudies

Optimization of phenol-chloroform RNA extraction.

Project description:Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis. •Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.

1000-01-01 | S-EPMC6031757 | BioStudies

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma.

Project description:Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.

1000-01-01 | S-EPMC3540853 | BioStudies

Quantification of total fatty acids in microalgae: comparison of extraction and transesterification methods.

Project description:Determination of microalgaes' fatty acid content is often done with chloroform and methanol according to the Bligh and Dyer extraction, though faster methods exist. A number of comparisons between the Bligh and Dyer and faster methods have resulted in contradicting data, possibly due to differences in algae used and the different versions of the Bligh and Dyer method applied. Here, various forms of direct-transesterification (D-TE) and two-step transesterification (2-TE), including three versions developed in our lab, are compared with the original Bligh and Dyer (Can J Biochem Physiol 37: 911-917, 1959) extraction and two modifications thereof (Lee et al. J AOAC Int 79:487-492, 1996, and our own acidified version) on microalgae with different cell walls: Isochrysis galbana, Nannochloropsis oculata, and Phaeodactylum tricornutum. In total, fatty acid extracts from 11 methods were separated and quantified by gas chromatography with mass spectrometry. Results show that, for N. oculata and P. tricornutum, methods based on chloroform-methanol underestimated the fatty acid content compared with the 2-TE and D-TE methods, which gave similar results. Moreover, D-TE methods are faster than chloroform-methanol methods and use chemicals that are less toxic. Of the D-TE methods, the ones using hydrochloric acid and sulfuric acid recovered the most fatty acids, while boron trifluoride recovered slightly less. The main qualitative difference between the fatty acids recovered was that the chloroform-methanol methods recovered less saturated fatty acids in P. tricornutum.

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An efficient modified method for plant leaf lipid extraction results in improved recovery of phosphatidic acid.

Project description:Lipidomics plays an important role in understanding plant adaptation to different stresses and improving our knowledge of the genes underlying lipid metabolism. Lipidomics involves lipid extraction, sample preparation, mass spectrometry analysis, and data interpretation. One of the practical challenges for large-scale lipidomics studies on plant leaves is the requirement of an efficient and rapid extraction method.A single-extraction method with a polar solvent mixture gives results comparable to a widely used, multi-extraction method when tested on both Arabidopsis thaliana and Sorghum bicolor leaf tissue. This single-extraction method uses a mixture of 30 parts chloroform, 25 parts isopropanol, 41.5 parts methanol, and 3.5 parts water (v/v/v/v) and a 24-h extraction time. Neither inclusion of ammonium acetate nor inclusion of acetic acid increased extraction efficiency.The extract produced by this method can be used for analysis by mass spectrometry without a solvent evaporation step. The amount of lipid extracted, including phosphatidic acid, is comparable to widely used, more labor-intensive methods. The single-extraction protocol is less laborious, reducing the potential for human error.

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Optimized DNA extraction and purification method for characterization of bacterial and fungal communities in lung tissue samples.

Project description:Human lungs harbor a scarce microbial community, requiring to develop methods to enhance the recovery of nucleic acids from bacteria and fungi, leading to a more efficient analysis of the lung tissue microbiota. Here we describe five extraction protocols including pre-treatment, bead-beating and/or Phenol:Chloroform:Isoamyl alcohol steps, applied to lung tissue samples from autopsied individuals. The resulting total DNA yield and quality, bacterial and fungal DNA amount and the microbial community structure were analyzed by qPCR and Illumina sequencing of bacterial 16S rRNA and fungal ITS genes. Bioinformatic modeling revealed that a large part of microbiome from lung tissue is composed of microbial contaminants, although our controls clustered separately from biological samples. After removal of contaminant sequences, the effects of extraction protocols on the microbiota were assessed. The major differences among samples could be attributed to inter-individual variations rather than DNA extraction protocols. However, inclusion of the bead-beater and Phenol:Chloroform:Isoamyl alcohol steps resulted in changes in the relative abundance of some bacterial/fungal taxa. Furthermore, inclusion of a pre-treatment step increased microbial DNA concentration but not diversity and it may contribute to eliminate DNA fragments from dead microorganisms in lung tissue samples, making the microbial profile closer to the actual one.

2020-01-01 | S-EPMC7562954 | BioStudies

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