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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
Published on: March 23, 2020
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Unexpected trypsin cleavage at ubiquitinated lysines
Analytical Chemistry
|July 17, 2015
Summary
Unexpected tryptic cleavage occurs at Lys48 residues in polyubiquitin chains. This results in unusual peptide fragments with a glycinylglycinyl-lysine terminus, observed across multiple trypsin sources.
Area of Science:
- Biochemistry
- Proteomics
- Molecular Biology
Background:
- Polyubiquitin chains are crucial for protein degradation and signaling.
- Tryptic digestion is a standard method for analyzing ubiquitin chain structures.
- Lysine 48 (K48) linkages are a common signal for proteasomal degradation.
Purpose of the Study:
- To investigate unexpected tryptic cleavage sites in polyubiquitin chains.
- To characterize the peptide products resulting from the digestion of various polyubiquitin chain types.
- To identify the cause of aberrant cleavage at K48-linked residues.
Main Methods:
- Analysis of tryptic digestion products from seven lysine-linked ubiquitin dimers.
- Characterization of three specific ubiquitin trimers (linear Ub-(48)Ub-(48)Ub, Ub-(63)Ub-(63)Ub, and branched [Ub]2-(6,48)Ub).
- Mass spectrometry-based analysis of peptide fragments.
Main Results:
- Observed unexpected tryptic cleavage at modified K48 residues in polyubiquitin chains.
- Identified peptide products with an unusual ε-glycinylglycinyl-Lys carboxyl terminus at K48 linkages.
- This aberrant cleavage was consistent across trypsin from three different commercial suppliers.
Conclusions:
- Tryptic digestion is not always predictable with modified K48-linked polyubiquitin.
- A novel cleavage mechanism involving initial cleavage at R74 in a distal ubiquitin is proposed.
- This finding has implications for ubiquitin chain analysis and proteomic studies.
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