Detailed Methodologies and Protocols

Righetti, Pier Giorgio; Boschetti, Egisto

doi:10.1016/b978-0-12-401734-4.00008-7

Cited by 6 publications

(3 citation statements)

References 94 publications

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“…A further increase in the number of identified/quantified proteins should be possible by, e.g., increasing the sample concentration or by pretreatment of the sample with combinatorial peptide ligand libraries (CPLL). In a comprehensive study applying the CPLL technology in combination with nanoLC–MS, Di Girolamo et al found an additional set of 440 proteins in yeast which were not detected in the untreated sample (total number of proteins found, 1785). , When comparing his data with our results, it was evident that in the untreated sample, 90% of the proteins were also found by the CE–MS approach, however, in the case of the CPLL treated sample the overlap was just 74%.…”

Section: Resultssupporting

confidence: 57%

Quantitative Proteomics Using Ultralow Flow Capillary Electrophoresis–Mass Spectrometry

et al. 2015

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In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE–MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE–MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE–MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE–MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE–MS analysis.

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Section: Resultssupporting

confidence: 57%

Quantitative Proteomics Using Ultralow Flow Capillary Electrophoresis–Mass Spectrometry

et al. 2015

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“…We next used ammonium sulfate to fractionate the cytosol from [ 3 H]cholesterol‐labelled upc2–1 cells to identify a fraction enriched in [ 3 H]cholesterol, and by implication, cytosolic SBPs. Ammonium sulfate is a lyotropic salt that is often used to salt out proteins; the functionality of ammonium sulfate‐precipitated proteins is usually fully recovered upon re‐solubilization in physiological buffers (Righetti & Boschetti, ). We found that treatment of the labelled cytosol with 50–60% ammonium sulfate resulted in precipitation of >80% of the radioactivity but <20% of the protein, a > 4‐fold enrichment of the SBP population (Figure b).…”

Section: Resultsmentioning

confidence: 99%

A PhotoClick cholesterol‐based quantitative proteomics screen for cytoplasmic sterol‐binding proteins in Saccharomyces cerevisiae

Chauhan¹,

Sere²,

Sokòl

et al. 2020

Yeast

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Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins.Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional-as yet unidentified-sterol carriers. To address this point, we hypothesized that sterol transport proteins are also sterol-binding proteins (SBPs), and used an in vitro chemoproteomic strategy to identify all cytosolic SBPs.We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologues revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol-binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking. K E Y W O R D Sclick chemistry, membrane, non-vesicular transport, proteomics, sterol

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“…In addition, precipitation with ammonium sulfate has no adverse effects on enzyme activity and does not denature protein structures. The protein functionality is fully recovered upon resolubilization in appropriate physiological buffers ( Righetti and Boschetti, 2013 ). Typically, TGM catalyzed transamidation between glutamine and lysine residues that can lead to the formation of covalent side-chain bridges between protein units; in this sense, TGM’s function as nature’s catalysts is to glue proteins together, and therefore to generate crosslinked supramolecular protein assemblies ( Lorand and Graham, 2003 ).…”

Section: Resultsmentioning

confidence: 99%

The Synthesis of (Magnetic) Crosslinked Enzyme Aggregates With Laccase, Cellulase, β-Galactosidase and Transglutaminase

Podrepšek

Knez

Leitgeb

2022

Front. Bioeng. Biotechnol.

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Immobilized enzymes have important aspects due to the fact that they possess higher stability, have the possibility to be easily removed from the reaction mixture, and are much easier to use when compared to free enzymes. In this research, the enzymes laccase, cellulase, β-galactosidase (β-gal), and transglutaminase (TGM) were immobilized by two different methods: crosslinked enzyme aggregates (CLEAs) and magnetic crosslinked enzyme aggregates (mCLEAs). The processes for CLEAs and mCLEAs preparation with different enzymes have been optimized, where the aim was to achieve the highest possible relative activity of the immobilized enzyme. The optimal conditions of the synthesis of CLEAs in mCLEAs are described, thus emphasizing the difference between the two types of immobilization based on different enzymes. This comparative study, which represents the synthesis of crosslinked enzyme aggregates using different enzymes, has not been performed so far. Moreover, the obtained activity of CLEAs and mCLEAs is presented, which is important for further use in different biocatalytic processes. Specifically, of a higher importance is the selection of enzymes involved in immobilization, as they belong to the three different most applicable enzymes (oxidoreductases, hydrolases, and transferases). The study confirmed that the resulting activity of the immobilized enzyme and the optimization of enzyme immobilization depended on the type of the enzyme. Moreover, the prepared CLEAs and mCLEAs were exposed to the supercritical carbon dioxide (scCO2) at different pressures to determine the effect of scCO2 on enzyme activity in immobilized form. Additionally, to demonstrate the reuse and stability of the immobilized enzyme, the stability and reusability tests of CLEAs and mCLEAs were performed. The catalytic performance of immobilized enzyme was tested, where the catalytic efficiency and long-term operational stability of mCLEAs were obviously superior to those of CLEAs. However, the higher activity observed for CLEAs compared to mCLEAs suggests a significant effect of magnetic nanoparticles in the stabilization of an enzyme crosslinked aggregate structure.

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Detailed Methodologies and Protocols

Cited by 6 publications

References 94 publications

Quantitative Proteomics Using Ultralow Flow Capillary Electrophoresis–Mass Spectrometry

Quantitative Proteomics Using Ultralow Flow Capillary Electrophoresis–Mass Spectrometry

A PhotoClick cholesterol‐based quantitative proteomics screen for cytoplasmic sterol‐binding proteins in Saccharomyces cerevisiae

The Synthesis of (Magnetic) Crosslinked Enzyme Aggregates With Laccase, Cellulase, β-Galactosidase and Transglutaminase

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