Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Doellinger J

PROVIDER: S-EPMC6944244 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Publications

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

Doellinger Joerg J Schneider Andy A Hoeller Marcell M Lasch Peter P

Molecular & cellular proteomics : MCP 20191121 1

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 be ...[more]

PMID: 31754045

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Project description:Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry.DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles.We describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.

Dataset Information

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

Publications

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

Similar Datasets

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