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Filter-aided sample preparation depends on filter unit shape

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Pancreatic Islet, Epithelial Cell, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Chiara guerrera

LAB HEAD: Ida Chiara Guerrera

PROVIDER: PXD003400 | Pride | 2020-11-05

REPOSITORIES: Pride

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			Action	DRS
	20141215_JoLI_Eth_100_n1_1431-01.msf	Msf
	20141215_JoLI_Eth_100_n1_1431.raw	Raw
	20141215_JoLI_Eth_100_n2_1432-01.msf	Msf
	20141215_JoLI_Eth_100_n2_1432.raw	Raw
	20141215_JoLI_Eth_100_n3_1433-01.msf	Msf

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Project description:The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. In our study, cells need to be exposed with same experimental cellular condition, semi lethal (IC50), respectively. In the case of SDS (CAS; 151-21-3), the exposure dose was decided as 0.01% by growth curve with continuously diluted exposure. SDS is an anionic surfactant that is suspected to be gastrointestinal or liver toxicant. // Toxicity of anionic detergents determined by Saccharomyces cerevisiae microarray analysis: Sodium n-dodecyl benzene sulfonate (LAS) and sodium dodecyl sulfate (SDS) are popular anionic detergents (surfactants) that are used worldwide and the toxicities of these chemicals have been characterized. We applied these chemicals in a DNA microarray bioassay and determined that the microarray data reflects previous findings and also provides some new information about anionic detergent toxicity. The mRNA expression profiles suggest that LAS and SDS cause damage to membranes and alterations in carbon metabolism, and induce the oxidative stress response. We also found that LAS and SDS induce the pleiotropic drug-resistance network, and that LAS and SDS may be pumped out of yeast cells by this network. Hierarchical clustering of the expression profiles showed that LAS and SDS cause similar features of toxicity and that the toxicity is similar to that of capsaicin but different from that of cadmium and mercury. Keywords: stress response

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Filter-aided sample preparation depends on filter unit shape

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