Sortase-mediated enrichment of ubiquitinated proteins from complex samples

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Protein ubiquitination is crucial for cellular signaling and protein regulation.
• Current methods for detecting ubiquitination, often antibody-dependent, struggle with non-canonical sites.
• There is a need for accessible and efficient techniques to identify ubiquitinated proteins.

Purpose of the Study:

• To develop a novel strategy for the detection of protein ubiquitination using sortase.
• To enable the enrichment and identification of ubiquitinated proteins, including non-canonical modifications.
• To provide an accessible tool for proteomics researchers studying ubiquitination.

Main Methods:

• Utilized sortase, a bacterial transpeptidase, to selectively tag ubiquitination sites with a biotin-modified peptide.
• Demonstrated the modification of N-terminal diglycine remnants in digested proteins.
• Applied the method to recombinant proteins, including Ubc13, for proof of concept.
• Investigated elution strategies using guanidinium or sortase reversibility.
• Incorporated a trypsin-labile bifunctional peptide to streamline the workflow.

Main Results:

• Successfully demonstrated selective biotinylation of ubiquitination sites via sortase.
• Identified a site of autoubiquitination in the E2 conjugating enzyme Ubc13.
• Showcased efficient peptide enrichment and elution using established or reversible methods.
• Validated the utility of a trypsin-labile peptide for improved downstream processing.

Conclusions:

• The sortase-mediated biotinylation strategy offers an accessible approach for detecting protein ubiquitination.
• This method facilitates the identification of both canonical and non-canonical ubiquitination sites.
• The developed technique has the potential to advance proteomics research by improving the detection of ubiquitinated proteins.