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

ABSTRACT: 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).

SUBMITTER: Wisniewski JR

PROVIDER: S-EPMC6811893 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Sensitivity and protein digestion course of proteomic Filter Aided Sample Preparation

Project description:1. Sensitivity of FASP was tested using SDS lysates from HeLa cells and mouse brain. Peptides were analyzed using a QExactive HF-X instrument. We found that FASP allows effective processing samples at the low and sub-microgram range. 2. 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. The results show that short digestion times apparently do not affect the number of identified proteins.

2019-11-12 | PXD014288 | Pride

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Filter-aided sample preparation depends on filter unit shape

Project description:One of the main goals of shotgun proteomics studies is to identify by mass spectrometry as many proteins as possible, often from low-protein samples, in order to help answer important biological questions. All experimental steps need to be optimized to achieve a thorough proteomic analysis. One of the most important is an optimal extraction and solubilization of proteins through the use of detergents, chaotropes, and reducing agents. Although very efficient in solubilizing membrane proteins, the presence of these detergents, such as SDS, negatively affects the LC/MS analysis and limits protein identifications, and therefor they need to be removed before LC-MS analysis. Filter-aided sample preparation (FASP) is a generally accepted method for removal of detergents and chaotropes used for protein extraction as well as non-proteinaceous compounds such as salts, nucleic acids and lipids. Another critical aspect of proteomic analyses is the starting amount of protein in the sample. There is still little information about the efficacy of FASP method for protein-limited samples. The aim of this study was to compare two methods for SDS removal, ethanol precipitation versus FASP and we evaluate the effectiveness of FASP method using different filtration devices, as well as RIPA (0.1% SDS) and high-SDS (2%SDS) lysis buffers applied to low (1µg) and high (10-100µg) protein amounts.

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Formaldehyde derivatization, a Side Reaction During Filter-Aided Sample Preparation

Project description:The filter-aided sample preparation (FASP) method has been commonly used for proteomic sample preparation due to its high efficiency and convenience. Herein, we report an overlooked side reaction during FASP method, resulting in a mass increment of 12 Da. We confirmed that the side reaction is caused by formaldehyde released from the spin filter and devel-oped a pretreatment step to reduce the side reaction. We further examined spin filters from different vendors and offered suggestions for different experimental purpose. Our findings would benefit the deep screening of labeling proteomics analy-sis and offer a new direction to improve performance of filter devices used for different biological and proteomics studies.

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

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

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