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Protease-resistant streptavidin for interaction proteomics.

ABSTRACT: Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow.

SUBMITTER: Rafiee MR

PROVIDER: S-EPMC7218406 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Protease-resistant streptavidin for interaction proteomics.

Rafiee Mahmoud-Reza MR Sigismondo Gianluca G Kalxdorf Mathias M Förster Laura L Brügger Britta B Béthune Julien J Krijgsveld Jeroen J

Molecular systems biology 20200501 5

Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biot ...[more]

PMID: 32400114

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Protease-resistant streptavidin for interaction proteomics

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2020-05-27 | PXD016576 | Pride

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A protease for 'middle-down' proteomics.

Project description:We developed a method for restricted enzymatic proteolysis using the outer membrane protease T (OmpT) to produce large peptides (>6.3 kDa on average) for mass spectrometry-based proteomics. Using this approach to analyze prefractionated high-mass HeLa proteins, we identified 3,697 unique peptides from 1,038 proteins. We demonstrated the ability of large OmpT peptides to differentiate closely related protein isoforms and to enable the detection of many post-translational modifications.

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Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs.

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Addressing the Protease Bias in Quantitative Proteomics.

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Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry.

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| S-EPMC5716544 | biostudies-literature

Toward Comprehensive Plasma Proteomics by Orthogonal Protease Digestion.

Project description:Rapid and consistent protein identification across large clinical cohorts is an important goal for clinical proteomics. With the development of data-independent technologies (DIA/SWATH-MS), it is now possible to analyze hundreds of samples with great reproducibility and quantitative accuracy. However, this technology benefits from empirically derived spectral libraries that define the detectable set of peptides and proteins. Here, we apply a simple and accessible tip-based workflow for the generation of spectral libraries to provide a comprehensive overview on the plasma proteome in individuals with and without active tuberculosis (TB). To boost protein coverage, we utilized nonconventional proteases such as GluC and AspN together with the gold standard trypsin, identifying more than 30,000 peptides mapping to 3309 proteins. Application of this library to quantify plasma proteome differences in TB infection recovered more than 400 proteins in 50 min of MS acquisition, including diagnostic Mycobacterium tuberculosis (Mtb) proteins that have previously been detectable primarily by antibody-based assays and intracellular proteins not previously described to be in plasma.

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Hydrophilic interaction liquid chromatography (HILIC) in proteomics.

Project description:In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications. [figure: see text]

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How the biotin-streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer.

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| S-EPMC3062853 | biostudies-literature

CIG-P: Circular Interaction Graph for Proteomics.

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