scholarly journals Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics based on Acid Extraction

2018 ◽  
Author(s):  
Joerg Doellinger ◽  
Andy Schneider ◽  
Marcell Hoeller ◽  
Peter Lasch
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Spectrometric Analysis ◽  
Acid Extraction ◽  
Sample Processing ◽  
Special Equipment ◽  
One Pot ◽  
Tissue Samples ◽  
Main Challenge ◽  
Speed Performance

SUMMARYMass spectrometry is the method of choice for deep and comprehensive analysis of proteomes and has become a key technology to support the progress in life science and biomedicine. However, sample preparation in proteomics is not standardized and contributes to a lack of reproducibility. The main challenge is to extract all proteins in a manner that enables efficient digestion into peptides and is compatible with subsequent mass spectrometric analysis. Current methods are based on the idea of removing detergents or chaotropic agents during sample processing, which are essential for protein extraction but interfere with digestion and LC-MS. These multi-step preparations are prone to losses, biases and contaminations, while being time-consuming and labor-intensive.We report a universal detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which is based on a simple three-step procedure, acidification, neutralization and digestion. SPEED is a one-pot method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction. SPEED-based sample processing is highly reproducible, provides exceptional peptide yields and enables preparation even of tissue samples with less than 15 min hands-on time and without any special equipment. Evaluation of SPEED performance revealed, that the number of quantified proteins and the quantitative reproducibility are superior compared to the well-established sample processing protocols FASP, ISD-Urea and SP3 for various sample types, including human cells, bacteria and tissue, even at low protein starting amounts.

scholarly journals Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid Extraction

2019 ◽  
Vol 19 (1) ◽  
pp. 209-222 ◽  
Author(s):  
Joerg Doellinger ◽  
Andy Schneider ◽  
Marcell Hoeller ◽  
Peter Lasch
Keyword(s):  
Sample Preparation ◽  
Quantitative Proteomics ◽  
Protein Extraction ◽  
Spectrometric Analysis ◽  
Acid Extraction ◽  
Universal Method ◽  
Proteome Coverage ◽  
Agent Based ◽  
Main Challenge ◽  
Sample Dissolution

The main challenge of bottom-up proteomic sample preparation is to extract proteomes in a manner that enables efficient protein digestion for subsequent mass spectrometric analysis. Today's sample preparation strategies are commonly conceptualized around the removal of detergents, which are essential for extraction but strongly interfere with digestion and LC-MS. These multi-step preparations contribute to a lack of reproducibility as they are prone to losses, biases and contaminations, while being time-consuming and labor-intensive. We report a detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which consists of three mandatory steps, acidification, neutralization and digestion. SPEED is a universal method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction by complete sample dissolution. The protocol is highly reproducible, virtually loss-less, enables very rapid sample processing and is superior to the detergent/chaotropic agent-based methods FASP, ISD-Urea and SP3 for quantitative proteomics. SPEED holds the potential to dramatically simplify and standardize sample preparation while improving the depth of proteome coverage especially for challenging samples.

Development of a C12Im-Cl-assisted method for rapid sample preparation in proteomic application

2021 ◽  
Author(s):  
Chang Liu ◽  
Xiaoxia Si ◽  
Shumei Yan ◽  
Xinyuan Zhao ◽  
Xiaohong Qian ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Extraction ◽  
Sample Processing ◽  
Processing Methods ◽  
Proteomic Analyses

Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods, including protein extraction and digestion, before MS remain as bottlenecks in the...

scholarly journals Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 566 ◽  
Author(s):  
Siemon Ng ◽  
Cassandra Braxton ◽  
Marc Eloit ◽  
Szi Feng ◽  
Romain Fragnoud ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Nucleic Acids ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Detection ◽  
Extraction Methods ◽  
Control Measures ◽  
Acid Extraction ◽  
Library Preparation ◽  
Sample Processing

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

scholarly journals An inexpensive semi-automated sample processing pipeline for cell-free RNA

2020 ◽  
Author(s):  
Mira N. Moufarrej ◽  
Stephen R. Quake
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Low Cost ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Sample Processing ◽  
Processing Pipeline ◽  
Liquid Handling ◽  
Sample Throughput ◽  
Automated Pipeline

AbstractDespite advances in automated liquid handling and microfluidics, preparing samples for RNA sequencing at scale generally requires expensive equipment, which is beyond the reach of many academic labs. Manual sample preparation remains a slow, expensive, and error-prone process. Here, we describe a low-cost, semi-automated pipeline to extract cell-free RNA (cfRNA) that increases sample throughput by 12-fold while reducing time spent and cost by nearly 11-fold and 3-fold respectively. This pipeline is generalizable for many nucleic acid extraction applications, thereby increasing the scale of studies, which can be performed in small research labs.

scholarly journals All-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay protocol for SARS-CoV-2 detection

2020 ◽  
Author(s):  
Xiong Ding ◽  
Kun Yin ◽  
Ziyue Li ◽  
Rajesh V. Lalla ◽  
Maroun M. Sfeir ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Fluorescence Detection ◽  
Visual Detection ◽  
Imaging System ◽  
Point Of Care ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
One Pot ◽  
Reaction Systems ◽  
Assay Protocol

Abstract This protocol presents the all-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay to ultra-sensitively and visually detect SARS-CoV-2. The procedure of AIOD-CRISPR assay typically consists of three parts including sample preparation, AIOD-CRISPR reaction, and fluorescence detection. Sample preparation involves the synthetic RNA preparation and the nucleic acid extraction from SARS-CoV-2 samples. The prepared nucleic acids were then added into the AIOD-CRISPR reaction systems as templates, followed by incubation at 37°C for 20-40 min. After incubation, visual detection was immediately conducted by placing the tubes in a portable LED blue transilluminator (Maestrogen UltraSlim) or the ChemiDoc™ MP Imaging System (Bio-Rad) with its built-in UV channel. In addition to endpoint visual detection, real-time fluorescence detection was also available for AIOD-CRISPR assay. This protocol is helpful for applying AIOD-CRISPR assay to rapid, sensitive, one-pot point-of-care SARS-CoV-2 detection.

scholarly journals Methodological Approaches to DNA Authentication of Foods, Wines and Raw Materials for Their Production

Foods ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 595
Author(s):  
Aram G. Galstyan ◽  
Vladislav K. Semipyatniy ◽  
Irina Yu. Mikhailova ◽  
Khamid Kh. Gilmanov ◽  
Alana V. Bigaeva ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Materials ◽  
Direct Sequencing ◽  
Genetic Identification ◽  
Proteinase K ◽  
Vitis Vinifera L ◽  
Acid Extraction ◽  
Plant Component ◽  
Sensitivity Specificity

DNA authentication of wines is a process of verifying their authenticity by genetic identification of the main plant component. The sample preparation of experimental and commercial wines was carried out by precipitation of wine debris by centrifugation with preliminary exposure with precipitators and co-precipitators, including developed macro- and micro- volume methods applicable to white or red wines, using polyvinylpyrrolidone as a co-precipitator. Addition of 2-mercaptoethanol and proteinase K to the lysing solution made it possible to adapt the technology for DNA extraction from the precipitated wine debris. The additionally tested technique of DNA extraction from wine debris by dimethyl sulfoxide (DMSO) lysis had fewer stages and, consequently, a lower risk of contamination. The results of further testing of one of the designed primer pairs (UFGT-F1 and UFGT-R1) in conjunction with the tested methods of wine material sample preparation and nucleic acid extraction, showed the advantage in the given set of oligonucleotides over previously used ones in terms of sensitivity, specificity and reproducibility. The developing strategy for genetic identification of grape varieties and DNA authentication of wines produced from them based on direct sequencing of polymerase chain reaction (PCR) products is implemented by interpreting the detected polymorphic positions of variable Vitis vinifera L. UFGT gene locus with distribution and split into 13 UFGT gene-associated groups.

Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System v2

2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Gram Negative ◽  
Protein Quantitation ◽  
System P ◽  
Automated Protocol ◽  
Chloroform Methanol ◽  
Extract Protein

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System

scholarly journals Proteomics, Holm Oak (Quercus ilex L.) and Other Recalcitrant and Orphan Forest Tree Species: How do They See Each Other?

2019 ◽  
Vol 20 (3) ◽  
pp. 692 ◽  
Author(s):  
María-Dolores Rey ◽  
María Castillejo ◽  
Rosa Sánchez-Lucas ◽  
Victor Guerrero-Sanchez ◽  
Cristina López-Hidalgo ◽  
...  
Keyword(s):  
Tree Species ◽  
Quercus Ilex ◽  
Protein Extraction ◽  
Forest Tree ◽  
Plant Biology ◽  
Spectrometric Analysis ◽  
Full Potential ◽  
Proteomics Data ◽  
Forest Tree Species ◽  
Complementary Techniques

Proteomics has had a big impact on plant biology, considered as a valuable tool for several forest species, such as Quercus, Pines, Poplars, and Eucalyptus. This review assesses the potential and limitations of the proteomics approaches and is focused on Quercus ilex as a model species and other forest tree species. Proteomics has been used with Q. ilex since 2003 with the main aim of examining natural variability, developmental processes, and responses to biotic and abiotic stresses as in other species of the genus Quercus or Pinus. As with the progress in techniques in proteomics in other plant species, the research in Q. ilex moved from 2-DE based strategy to the latest gel-free shotgun workflows. Experimental design, protein extraction, mass spectrometric analysis, confidence levels of qualitative and quantitative proteomics data, and their interpretation are a true challenge with relation to forest tree species due to their extreme orphan and recalcitrant (non-orthodox) nature. Implementing a systems biology approach, it is time to validate proteomics data using complementary techniques and integrate it with the -omics and classical approaches. The full potential of the protein field in plant research is quite far from being entirely exploited. However, despite the methodological limitations present in proteomics, there is no doubt that this discipline has contributed to deeper knowledge of plant biology and, currently, is increasingly employed for translational purposes.

scholarly journals Comparative Proteomic Analysis of Advanced Ovarian Cancer Tissue to Identify Potential Biomarkers of Responders and Nonresponders to First-Line Chemotherapy of Carboplatin and Paclitaxel

2016 ◽  
Vol 8 ◽  
pp. BIC.S35775 ◽  
Author(s):  
Urmila Sehrawat ◽  
Ruchika Pokhriyal ◽  
Ashish Kumar Gupta ◽  
Roopa Hariprasad ◽  
Mohd Imran Khan ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Advanced Ovarian Cancer ◽  
Spectrometric Analysis ◽  
Cancer Tissue ◽  
Chemotherapy Response ◽  
Tissue Samples ◽  
Post Surgery ◽  
Dismal Prognosis ◽  
Apoptotic Activity ◽  
Potential Biomarkers

Conventional treatment for advanced ovarian cancer is an initial debulking surgery followed by chemotherapy combination of carboplatin and paclitaxel. Despite initial high response, three-fourths of these women experience disease recurrence with a dismal prognosis. Patients with advanced-stage ovarian cancer who underwent cytoreductive surgery were enrolled and tissue samples were collected. Post surgery, these patients were started on chemotherapy and followed up till the end of the cycle. Fluorescence-based differential in-gel expression coupled with mass spectrometric analysis was used for discovery phase of experiments, and real-time polymerase chain reaction, Western blotting, and pathway analysis were performed for expression and functional validation of differentially expressed proteins. While aldehyde reductase, hnRNP, cyclophilin A, heat shock protein-27, and actin are upregulated in responders, prohibitin, enoyl-coA hydratase, peroxiredoxin, and fibrin-β are upregulated in the nonresponders. The expressions of some of these proteins correlated with increased apoptotic activity in responders and decreased apoptotic activity in nonresponders. Therefore, the proteins qualify as potential biomarkers to predict chemotherapy response.

Smartphone integrated optoelectrowetting (SiOEW) for on-chip sample processing and microscopic detection of water quality

Lab on a Chip ◽  
2018 ◽  
Vol 18 (3) ◽  
pp. 532-539 ◽  
Author(s):  
Dongyue Jiang ◽  
Seunguk Lee ◽  
Sung Woo Bae ◽  
Sung-Yong Park
Keyword(s):  
Water Quality ◽  
Sample Preparation ◽  
Low Cost ◽  
Sample Processing ◽  
Microscopic Detection ◽  
Chip Sample ◽  
On Chip

We present a smartphone integrated optoelectrowetting (SiOEW) device as a low-cost, portable tool for on-chip sample preparation and microscopic detection of water quality.

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