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SCASP: A Simple and Robust SDS-Aided Sample Preparation Method for Proteomic Research.

ABSTRACT: SDS is widely used in sample preparation for proteomic research. However, SDS is incompatible with LC and electrospray ionization. SDS depletion is therefore required ahead of LC-MS analysis. Most of current SDS removal strategies are time consuming, laborious, and have low reproducibility. Here, we describe a method, SDS-cyclodextrin (CD)-assisted sample preparation, by which CD can bind to SDS and form CD-SDS complexes in solutions, allowing for direct tryptic digestion. We demonstrate that SDS-CD-assisted sample preparation is a simple, fast, and robust SDS-based sample preparation method for proteomics application.

SUBMITTER: Gan G

PROVIDER: S-EPMC7970136 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

Project description:We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists.

| S-EPMC4409837 | biostudies-literature

Datasets: Sensitivity and protein digestion course of proteomic Filter Aided Sample Preparation.

Project description:Sensitivity of FASP was tested using SDS lysates from HeLa cells and mouse brain. Peptides were analyzed using a QExactive HF-X instrument. Whole cell lysates of Hela cells were processed with FASP using single or double, consecutive or successive, digestion with LysC or trypsin. The generated peptides were analyzed using a LTQ-Orbitrap mass spectrometer. These datasets accompany "Filter Aided Sample Preparation - A Tutorial" (Wiśniewski, 2019).

| S-EPMC6811893 | biostudies-literature

ZASP：A Robust Method for Proteomics Sample Preparation

Project description:ZASP can effectively remove SDS, SDC, TritonX-100 and other denaturing reagents. • ZASP has short precipitation time, high protein recovery and identification number. • Compared with acetone and SP3 precipitation method, ZASP has remarkable performance. • ZASP is particularly suitable for complex samples, simplifying the sample processing process and increasing the number of protein identifications.

2023-05-16 | PXD042276 |

Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis.

Project description:Mass spectrometry-based urinary proteomics is one of the most attractive strategies to discover proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of urological diseases involving the kidney, prostate, and bladder; however, interfering compounds found in urine necessitate sample preparation strategies that are currently not suitable for urinary proteomics in the clinical setting. Herein, we describe the C4-tip method, comprising a simple, automated strategy utilizing a reverse-phase resin tip-based format and "on-tip" digestion to examine the urine proteome. We first determined the optimal conditions for protein isolation and protease digestion on the C4-tip using the standard protein bovine fetuin. Next, we applied the C4-tip method to urinary proteomics, identifying a total of 813 protein groups using LC-MS/MS, with identified proteins from the C4-tip method displaying a similar distribution of gene ontology (GO) cellular component assignments compared to identified proteins from an ultrafiltration preparation method. Finally, we assessed the reproducibility of the C4-tip method, revealing a high Spearman correlation R-value for shared proteins identified across all tips. Together, we have shown the C4-tip method to be a simple, robust method for high-throughput analysis of the urinary proteome by mass spectrometry in the clinical setting.

| S-EPMC6512789 | biostudies-literature

Ultrasonic-based filter aided sample preparation as the general method to sample preparation in proteomics

Project description:In this work, we propose a new high-throughput ultrafast method for large scale proteomics approaches by speeding the classic filter aided sample preparation protocol, FASP. The new US-FASP method matches the analytical minimalism roles as time, cost, sample requirement, reagent consumption, energy requirements and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all the advantages of the filter aided sample preparation protocol are maintained.

2020-06-08 | PXD018360 | Pride

Miniaturized Filter-Aided Sample Preparation (MICRO-FASP) Method for High Throughput, Ultrasensitive Proteomics Sample Preparation Reveals Proteome Asymmetry in <i>Xenopus laevis</i> Embryos.

Project description:We report a miniaturized filter aided sample preparation method (micro-FASP) for low-loss preparation of submicrogram proteomic samples. The method employs a filter with ∼0.1 mm2 surface area, reduces the total volume of reagents to <10 μL, and requires only two sample transfer steps. The method was used to generate 25 883 unique peptides and 3069 protein groups from 1000 MCF-7 cells (∼100 ng protein content), and 13 367 peptides and 1895 protein groups were identified from 100 MCF-7 cells (∼10 ng protein content). Single blastomeres from Xenopus laevis embryos at the 50-cell stage (∼200 ng yolk free protein/blastomere) generated 20 943 unique peptides and 2597 protein groups; the proteomic profile clearly differentiated left and right blastomeres and provides strong support for models in which this asymmetry is established early in the embryo. The parallel processing of 12 samples demonstrates reproducible label free quantitation of 1 μg protein homogenates.

| S-EPMC7931810 | biostudies-literature

Simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomics applications.

Project description:Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for biological sample extraction; however, the presence of this reagent in samples challenges LC-MS-based proteomics analyses because it can interfere with reversed-phase LC separations and electrospray ionization. This study reports a simple SDS-assisted proteomics sample preparation method facilitated by a novel peptide-level SDS removal step. In an initial demonstration, SDS was effectively (>99.9%) removed from peptide samples through ion substitution-mediated DS(-) precipitation using potassium chloride (KCl), and excellent peptide recovery (>95%) was observed for <20 ?g of peptides. Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage obtained for both mammalian tissues and bacterial samples was comparable to or better than that obtained for the same sample types prepared using standard proteomics preparation methods and analyzed using LC-MS/MS. These results suggest the SDS-assisted protocol is a practical, simple, and broadly applicable proteomics sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.

| S-EPMC3310275 | biostudies-literature

A Robust Method for Sample Preparation of Gastrointestinal Stromal Tumour for LC/MS Untargeted Metabolomics.

Project description:Gastrointestinal stromal tumour has already been well explored at the genome level; however, little is known about metabolic processes occurring in the sarcoma. Sample preparation is a crucial step in untargeted metabolomics workflow, highly affecting the metabolome coverage and the quality of the results. In this study, four liquid-liquid extraction methods for the isolation of endogenous compounds from gastrointestinal stromal tumours were compared and evaluated. The protocols covered two-step or stepwise extraction with methyl-tert-butyl ether (MTBE) or dichloromethane. The extracts were subjected to LC-MS analysis by the application of reversed-phase and hydrophilic interaction liquid chromatography to enable the separation and detection of both polar and nonpolar analytes. The extraction methods were compared in terms of efficiency (total number of detected metabolites) and reproducibility. The method was based on the stepwise extraction with MTBE, methanol, and water proved to be the most reproducible, and thus, its robustness to fluctuations in experimental conditions was assessed employing Plackett-Burman design and hierarchical modelling. While most studied factors had no effect on the metabolite abundance, the highest coefficient value was observed for the volume of MTBE added during extraction. Herein, we demonstrate the application and the feasibility of the selected protocol for the analysis of gastrointestinal stromal tumour samples. The method selected could be considered as a reference for the best characterization of underlying molecular changes associated with complex tissue extracts of GIST.

| S-EPMC8400919 | biostudies-literature

Disclosing the molecular profile of the human amniotic mesenchymal stromal cell secretome by filter-aided sample preparation proteomic characterization.

Project description:BackgroundThe secretome of mesenchymal stromal cells isolated from the amniotic membrane (hAMSCs) has been extensively studied for its in vitro immunomodulatory activity as well as for the treatment of several preclinical models of immune-related disorders. The bioactive molecules within the hAMSCs secretome are capable of modulating the immune response and thus contribute to stimulating regenerative processes. At present, only a few studies have attempted to define the composition of the secretome, and several approaches, including multi-omics, are underway in an attempt to precisely define its composition and possibly identify key factors responsible for the therapeutic effect.MethodsIn this study, we characterized the protein composition of the hAMSCs secretome by a filter-aided sample preparation (FASP) digestion and liquid chromatography-high resolution mass spectrometry (LC-MS) approach. Data were processed for gene ontology classification and functional protein interaction analysis by bioinformatics tools.ResultsProteomic analysis of the hAMSCs secretome resulted in the identification of 1521 total proteins, including 662 unique elements. A number of 157 elements, corresponding to 23.7%, were found as repeatedly characterizing the hAMSCs secretome, and those that resulted as significantly over-represented were involved in immunomodulation, hemostasis, development and remodeling of the extracellular matrix molecular pathways.ConclusionsOverall, our characterization enriches the landscape of hAMSCs with new information that could enable a better understanding of the mechanisms of action underlying the therapeutic efficacy of the hAMSCs secretome while also providing a basis for its therapeutic translation.

| S-EPMC10683150 | biostudies-literature

iFASP: combining isobaric mass tagging with filter-aided sample preparation.

Project description:Careful, clean and controlled preparation of samples for mass spectrometry proteomics is crucial to obtain reproducible and reliable data. This is especially important when carrying out quantitative proteomics by chemical isobaric labeling (aka tandem mass tagging), since the differentially labeled samples are combined quite late during the sample processing. Addressing this need for robust and reliable sample processing for quantitative proteomics, we describe here iFASP, a simple protocol for combining isobaric mass tagging with the recently introduced filter-aided sample preparation (FASP) method. iFASP provides a quick, simple and effective method for obtaining clean samples, ensuring efficient digestion and providing excellent labeling yields for quantitative proteomics experiments. We have carried out our iFASP protocol using several highly complex Xenopus laevis egg and embryo lysates and compared the labeling yields and number of high-confidence peptide identifications to a standard in-solution digestion and labeling protocol. Although the labeling efficiency with both techniques is in the 99+% range, the number of peptides identified with a 1% false discovery rate and the corresponding number of quantified peptide spectral matches are as much as doubled with iFASP compared to the corresponding non-FASP-based method.

| S-EPMC4416645 | biostudies-literature

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