scholarly journals Solvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads

2021 ◽  
Author(s):  
Harvey E Johnston ◽  
Kranthikumar Yadav ◽  
Joanna M Kirkpatrick ◽  
George S Biggs ◽  
David Oxley ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Magnetic Beads ◽  
Solid Phase ◽  
Low Cost ◽  
Contaminant Removal ◽  
Protein Capture ◽  
Protein Immobilisation ◽  
Significant Enrichment ◽  
Low Solubility ◽  
A Current

Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture proteins, with subsequently washes allowing contaminant removal. However, SP3 is dependent on effective protein immobilisation onto beads, risks losses during wash steps, and experiences a drop-off in protein recovery at higher protein inputs. Magnetic beads may also contaminate samples and instruments, and become costly for larger scale protein preparations. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3, omitting magnetic beads and employing brief centrifugation—either with or without low-cost inert glass beads—as the means of aggregated protein capture. SP4 recovered equivalent or greater protein yields for 1–5000 μg preparations and improved reproducibility (median protein R2 SP4 0.99 vs. SP3 0.97). Deep proteome profiling (n = 9,076) also demonstrated improved recovery by SP4 and a significant enrichment of membrane and low-solubility proteins vs. SP3. The effectiveness of SP4 was verified in three other labs, each confirming equivalent or improved proteome characterisation over SP3. This work suggests that protein precipitation is the primary mechanism of SP3, and reliance on magnetic beads presents protein losses, especially at higher concentrations and amongst hydrophobic proteins. SP4 represents an efficient and effective alternative to SP3, provides the option to omit beads entirely, and offers virtually unlimited scalability of input and volume—all whilst retaining the speed and universality of SP3.

Analysis of the Non-Specific Binding Proteins in the RNA Pull-Down Experiment

2020 ◽  
Vol 17 ◽  
Author(s):  
Yongmei Li ◽  
Baicai Yang ◽  
Yali Zhang ◽  
Kaiwen Hei ◽  
Mingming Xiao
Keyword(s):  
Sample Preparation ◽  
Binding Proteins ◽  
Magnetic Beads ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Solid Phase ◽  
Specific Binding ◽  
Mass Spectrometric ◽  
Spectrometric Analysis ◽  
Preparation Conditions

Background: To investigate the interactions between RNA and proteins is essential to understand how these macromolecule complexes exert their functions. RNA pull-down is a classic technique to enrich RNA binding proteins, however, a large number of non-specific binding proteins may be enriched during sample preparation, interfering with the downstream mass spectrometric analyses and also causing false positives. Objective: In this study we examined the background contaminates in RNA pull-down experiment using mass spectrometric analysis. Method Antisense MALAT1 was first synthesized using in vitro transcription and incubated with cellular proteins extracted from HepG2 cells. The non-specific binding proteins were isolated using streptavidin conjugated magnetic beads and separated on SDS-PAGE. Each gel lane was divided into nine bands and digested with trypsin for the downstream LC-MS/MS analyses. Results: 191 protein groups were identified as non-specific binding proteins in RNA pull-down samples. In addition, comparison between different sample preparation conditions showed that the level of background contaminates were mostly induced by the solid phase support and not affected by the studied RNA. In addition, using more stringent detergent and streptavidin magnetic beads with smaller size could reduce the amount of background interfering proteins. Conclusion: This study provides a reference to distinguish bona fide RNA interacting proteins from the background contaminants. The results also demonstrate that different sample preparation conditions have great impacts on the level of enriched background contaminates, shedding new light on the optimization of RNA pull-down experiment.

Innovative Solutions for the Isolation of Vitamins from Pharmaceutical and Biological Samples

2020 ◽  
Vol 16 ◽  
Author(s):  
Giovanni D’Orazio ◽  
Alessandra Gentili ◽  
Salvatore Fanali ◽  
Chiara Fanali ◽  
Chiara Dal Bosco
Keyword(s):  
Sample Preparation ◽  
Biological Samples ◽  
Solid Phase ◽  
Low Cost ◽  
Current Trend ◽  
Sample Processing ◽  
Processing Times ◽  
Innovative Solutions ◽  
Microextraction Techniques ◽  
Neoteric Solvents

BACKGROUND: Isolation of vitamins from complex matrices, such as pharmaceutical and biological samples, is a demanding task. Progresses in material science are revolutionizing sample preparation, leading to the development of more efficient, sensitive and selective analytical methods. In particular, the evolution of solid-phase extraction towards miniaturized techniques is closely related to the introduction of nanomaterials as new sorbents. Microextraction techniques, both solid-based and liquid-based, allow for the reduction of solvent and sample volumes, as well as the sample processing times. In many cases these procedures are also environmentally friendly. Moreover, the substitution of traditional organic solvents with the neoteric ones is another current trend in analytical chemistry. The aim of this review is to introduce readers to the innovative solutions available for the extraction of vitamins from matrices of different complexity. METHODS: Research papers in the literature of last five years are reviewed, providing a critical description of the most original and interesting sample preparation approaches devoted to vitamin isolation. RESULTS: Among the last 5 years literature concerning the analysis of vitamins in the clinical and pharmaceutical sectors, only 40% of works applied some innovative or green approaches to sample preparation. However, a general trend is the reduction of solvent volumes and sample processing times. CONCLUSION: The great potential of microextraction techniques based on nanomaterials and neoteric solvents has not been fully exploited yet in the clinical and pharmaceutical fields. Considering the high cost of carbon-based nanomaterials, it can be foreseen that the most promising solution for sample preparation in the near future are the liquid microextraction techniques based on neoteric solvents. Thanks to their low cost, environmental compatibility, simplicity, speediness and high enrichment factor, these extractive techniques are particularly attractive for vitamin analysis.

scholarly journals Miniaturized Sample Preparation Methods to Simultaneously Determine the Levels of Glycols, Glycol Ethers and Their Acetates in Cosmetics

Cosmetics ◽  
2021 ◽  
Vol 8 (4) ◽  
pp. 102
Author(s):  
Maria Celeiro ◽  
Laura Rubio ◽  
Carmen Garcia-Jares ◽  
Marta Lores
Keyword(s):  
Sample Preparation ◽  
Solid Phase ◽  
Low Cost ◽  
Direct Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Glycol Ethers ◽  
Ultrasound Assisted Extraction ◽  
Preparation Methods ◽  
Accuracy And Precision ◽  
Assisted Extraction

Two environmentally friendly methodologies based on ultrasound-assisted extraction (UAE) and micro-matrix solid-phase dispersion (µMSPD) followed by gas chromatography-mass spectrometry (GC-MS) analysis are proposed for the first time for the simultaneous analysis of 17 glycols, glycol ethers, and their acetates in cosmetics. These sample preparation approaches result in efficient and low-cost extraction while employing small amounts of sample, with a low consumption of reagents and organic solvents. The use of a highly polar column allows for the direct analysis of the obtained extracts by GC-MS without a previous derivatization step, drastically reducing the sample preparation time and residues and thus complying with green analytical chemistry (GAC) principles. Both the UAE and µMSPD methodologies were validated in terms of linearity, accuracy, and precision, providing satisfactory results. LODs were found to be lower than 0.75 µg g−1, allowing the determination of trace levels of the forbidden target compounds. Finally, the validated methodologies were applied to real cosmetics and personal care products, showing suitability, and providing a reliable and useful tool for cosmetics control laboratories.

To Eliminate Curtain Effect of FIB TEM Samples by a Combination of Sample Dicing and Backside Milling

2014 ◽  
Author(s):  
Jian-Shing Luo ◽  
Hsiu Ting Lee
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Low Cost ◽  
Ion Beam ◽  
Sample Preparation Method ◽  
Site Specific ◽  
Dual Beam ◽  
Transmission Electron

Abstract Several methods are used to invert samples 180 deg in a dual beam focused ion beam (FIB) system for backside milling by a specific in-situ lift out system or stages. However, most of those methods occupied too much time on FIB systems or requires a specific in-situ lift out system. This paper provides a novel transmission electron microscopy (TEM) sample preparation method to eliminate the curtain effect completely by a combination of backside milling and sample dicing with low cost and less FIB time. The procedures of the TEM pre-thinned sample preparation method using a combination of sample dicing and backside milling are described step by step. From the analysis results, the method has applied successfully to eliminate the curtain effect of dual beam FIB TEM samples for both random and site specific addresses.

Recent Trends in the Development of Green Microextraction Techniques for the Determination of Hazardous Organic Compounds in Wine

2019 ◽  
Vol 15 (7) ◽  
pp. 788-800 ◽  
Author(s):  
Natasa P. Kalogiouri ◽  
Victoria F. Samanidou
Keyword(s):  
Sample Preparation ◽  
Solid Phase Extraction ◽  
Organic Compounds ◽  
Method Development ◽  
Solid Phase ◽  
Stir Bar Sorptive Extraction ◽  
Phase Extraction ◽  
Microextraction Techniques ◽  
Hazardous Organic Compounds

Background:The sample preparation is the most crucial step in the analytical method development. Taking this into account, it is easily understood why the domain of sample preparation prior to detection is rapidly developing. Following the modern trends towards the automation, miniaturization, simplification and minimization of organic solvents and sample volumes, green microextraction techniques witness rapid growth in the field of food quality and safety. In a globalized market, it is essential to face the consumers need and develop analytical methods that guarantee the quality of food products and beverages. The strive for the accurate determination of organic hazards in a famous and appreciated alcoholic beverage like wine has necessitated the development of microextraction techniques.Objective:The objective of this review is to summarize all the recent microextraction methodologies, including solid phase extraction (SPE), solid phase microextraction (SPME), liquid-phase microextraction (LPME), dispersive liquid-liquid microextraction (DLLME), stir bar sorptive extraction (SBSE), matrix solid-phase dispersion (MSPD), single-drop microextraction (SDME) and dispersive solid phase extraction (DSPE) that were developed for the determination of hazardous organic compounds (pesticides, mycotoxins, colorants, biogenic amines, off-flavors) in wine. The analytical performance of the techniques is evaluated and their advantages and limitations are discussed.Conclusion:An extensive investigation of these techniques remains vital through the development of novel strategies and the implication of new materials that could upgrade the selectivity for the extraction of target analytes.

scholarly journals A Toolbox for the Determination of Nitroaromatic Explosives in Marine Water, Sediment, and Biota Samples on Femtogram Levels by GC-MS/MS

Toxics ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 60
Author(s):  
Tobias Hartwig Bünning ◽  
Jennifer Susanne Strehse ◽  
Ann Christin Hollmann ◽  
Tom Bötticher ◽  
Edmund Maser
Keyword(s):  
Sample Preparation ◽  
Freeze Drying ◽  
Solid Phase ◽  
Phase Extraction ◽  
Direct Extraction ◽  
Time Saving ◽  
Marine Samples ◽  
Volume Method ◽  
Biota Samples

To determine the amount of the explosives 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and its metabolites in marine samples, a toolbox of methods was developed to enhance sample preparation and analysis of various types of marine samples, such as water, sediment, and different kinds of biota. To achieve this, established methods were adapted, improved, and combined. As a result, if explosive concentrations in sediment or mussel samples are greater than 10 ng per g, direct extraction allows for time-saving sample preparation; if concentrations are below 10 ng per g, techniques such as freeze-drying, ultrasonic, and solid-phase extraction can help to detect even picogram amounts. Two different GC-MS/MS methods were developed to enable the detection of these explosives in femtogram per microliter. With a splitless injector, limits of detection (LODs) between 77 and 333 fg/µL could be achieved in only 6.25 min. With the 5 µL programmable temperature vaporization—large volume method (PTV-LVI), LODs between 8 and 47 fg/µL could be achieved in less than 7 min. The detection limits achieved by these methods are among the lowest published to date. Their reliability has been tested and confirmed by measuring large and diverse sample sets.

scholarly journals A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAs

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 468
Author(s):  
Anthony E. Jones ◽  
Nataly J. Arias ◽  
Aracely Acevedo ◽  
Srinivasa T. Reddy ◽  
Ajit S. Divakaruni ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Biosynthetic Pathway ◽  
Solid Phase ◽  
High Sensitivity ◽  
Short Chain ◽  
Extraction Column ◽  
Chain Acyl ◽  
Precision And Accuracy ◽  
Mass Spectrometry Mass Spectrometry

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.

scholarly journals Improvement of long-term cycling performance of high-nickel cathode materials by ZnO coating

2021 ◽  
Vol 10 (1) ◽  
pp. 210-220
Author(s):  
Fangfang Wang ◽  
Ruoyu Hong ◽  
Xuesong Lu ◽  
Huiyong Liu ◽  
Yuan Zhu ◽  
...  
Keyword(s):  
Electrochemical Performance ◽  
Lithium Ion Batteries ◽  
Solid Phase ◽  
Low Cost ◽  
Specific Capacity ◽  
High Capacity ◽  
Lithium Ion ◽  
Side Reaction ◽  
Chemical Route ◽  
High Nickel

Abstract The high-nickel cathode material of LiNi0.8Co0.15Al0.05O2 (LNCA) has a prospective application for lithium-ion batteries due to the high capacity and low cost. However, the side reaction between the electrolyte and the electrode seriously affects the cycling stability of lithium-ion batteries. In this work, Ni2+ preoxidation and the optimization of calcination temperature were carried out to reduce the cation mixing of LNCA, and solid-phase Al-doping improved the uniformity of element distribution and the orderliness of the layered structure. In addition, the surface of LNCA was homogeneously modified with ZnO coating by a facile wet-chemical route. Compared to the pristine LNCA, the optimized ZnO-coated LNCA showed excellent electrochemical performance with the first discharge-specific capacity of 187.5 mA h g−1, and the capacity retention of 91.3% at 0.2C after 100 cycles. The experiment demonstrated that the improved electrochemical performance of ZnO-coated LNCA is assigned to the surface coating of ZnO which protects LNCA from being corroded by the electrolyte during cycling.

scholarly journals Adsorption of copper ions on Magnolia officinalis residues after solid-phase fermentation with Phanerochaete chrysosporium

2019 ◽  
Vol 17 (1) ◽  
pp. 1173-1184 ◽  
Author(s):  
Fengyun Tao ◽  
Yangping Liu ◽  
Junliang Chen ◽  
Peng Wang ◽  
Qing Huo
Keyword(s):  
Chinese Medicine ◽  
Phanerochaete Chrysosporium ◽  
Solid Phase ◽  
Copper Ions ◽  
Removal Rate ◽  
Low Cost ◽  
Preparation Technique ◽  
Adsorption Enthalpy ◽  
Pseudo Second Order ◽  
Magnolia Officinalis

AbstractThe disposal of residues while manufacturing Chinese medicine has always been an issue that concerns pharmaceutical factories. Phanerochaete chrysosporium was inoculated into the residues of Magnolia officinalis for solid-phase fermentation to enzymatically hydrolyze the lignin in the residues and thus to improve the efficiency of removal of the copper ions from residues for the utilization of residues from Chinese medicine. With the increase in activities of lignin-degrading enzymes, especially during the fermentation days 6 to 9, the removal rate of copper ions using M. officinalis residues increased dramatically. The rate of removal reached the maximum on the 14th day and was 3.15 times higher than the initial value. The rate of adsorption of copper ions on the fermentation-modified M. officinalis residues followed the pseudo-second-order kinetics. The adsorption isotherms were consistent with the Freundlich models. The adsorption enthalpy was positive, indicating that it was endothermic and elevation in temperature was favorable to this adsorption process. The adsorption free energy was negative, implying the spontaneity of the process. The copper ions adsorbed could be effectively recovered using 0.2 M hydrochloric acid solution. After five successive cycles of adsorption-regeneration, the fermentation-modified M. officinalis residues exhibited a stable adsorption capacity and greater reusability. The M. officinalis residues fermented with P. chrysosporium are low-cost and environmentally friendly copper ions adsorbent, and this preparation technique realizes the optimum utilization of Chinese medicine residues.

Sign in / Sign up

Export Citation Format

Share Document