Dataset Information

Protein precipitation of diluted samples with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach

ABSTRACT: Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest especially due to the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in a case of very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH-MS yields which were compared with the results from the same sample without precipitation. With this study it was possible to conclude that, in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery, number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone when comparing to the original sample.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain

SUBMITTER: Cátia Santa

LAB HEAD: Bruno Manadas

PROVIDER: PXD003653 | Pride | 2016-04-25

REPOSITORIES: Pride

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Publications

Protein precipitation of diluted samples in SDS-containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach.

Santa Cátia C Anjo Sandra I SI Manadas Bruno B

Proteomics 20160523 13

Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (co ...[more]

PMID: 27094026

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Project description:We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein profiled the host proteomic alterations resulted by the infection of THP-1 macrophages with R. conorii and R. montanensis using a high throughput quantitative proteomics approach (SWATH-MS). Our results revealed that these two members of SFG Rickettsia with distinct pathogenicity attributes for humans, trigger differential proteomic signatures in macrophage-like cells. Although infection by both rickettsial species resulted in a lower abundance of enzymes of glycolysis and pentose phosphate pathway, the pathogenic R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid -oxidation and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii towards a metabolic signature of an M2-like (anti-inflammatory) activation program. Moreover, our results revealed that several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. Remarkably, R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to compensate the accumulation of misfolded proteins by increasing the ER protein folding capacity and subsequently restore host cell homeostasis. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage.

Dataset Information

Protein precipitation of diluted samples with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach

Publications

Protein precipitation of diluted samples in SDS-containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach.

Similar Datasets

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