Gg

Tryptic digestion

Signature GG-peptides: Mass addition of 114.043 Da Miscleavage at modified sites

polyUb chain linkages

Ubiquitination sites

Peptides for protein identification

FIGURE 30.3. Analysis of a ubiquitinated protein by trypsin digestion and MS. Trypsin is a specific endoprotease that cleaves the peptide bonds at the C-termini of Arg or Lys unless followed by a Pro residue. When a Ub conjugate is digested with trypsin, the resulting peptides (on the right-hand side, dashed lines) can be used for determining the identity of the sequence. The peptide containing -GG tag (in the middle, solid lines) is generated by three cleavage events marked by arrows and is used for identifying the ubiquitination site by MS/MS. In addition, the -GG peptide digested from the polyUb chain (on the left-hand side, dashed lines) is useful for examining the chain linkage.

the -GG peptides contain a missed tryptic cleavage at the modified Lys sites. Based on these features, the Ub modification sites can be identified by unique MS/MS spectra of the -GG peptides during database searching. However, some ULPs, such as Nedd8 and ISG15, generate the same -GG peptides after trypsin digestion, and therefore it is prerequisite to separate those proteins by prefractionation. Another caveat is the difficulty to map all ubiquitinated sites in isolated Ub conjugates by current "shotgun" MS, because of undersampling (i.e., only a fraction of peptides are analyzed by MS/MS when many peptides are co-eluted together), lack of sensitivity (i.e., the recovery and/or ionization efficiency of some peptides are too low to be detected), and incompatibility (i.e., -GG peptides are too long or too short for the used proteomics platform; or the quality of MS/MS spectra is not good enough for effective database matching and quality filtering).

3. Evaluate the status of ubiquitination by the shift of apparent MW. In GeLC-MS/MS analysis, the MW information can be obtained from the SDS gel, which can be used to derive a virtual Western blotting result for identified proteins. The MW of unmodified proteins can be calculated from their amino acid sequence or even measured by GeLC-MS/MS. The comparison between native forms and ubiquitination forms would verify the status of Ub modification and lead to estimation of the length of Ub chains (~8 kDa for Ub monomer, ~16 kDa for di-Ub or double monoUb on two modification sites, and so on).

4. Affirm Ub conjugates of interest individually by immunoprecipitation. This independent approach has low throughput and can be used for analyzing selected proteins. The protein (or tagged version) is normally immunoisolated and probed with antibodies against the protein itself and Ub. Ubiquitination is often verified by the detection of mass shift and HMW smear. Highly stringent condition (e.g., SDS) should be included to minimize co-immunoprecipitation of other ubiquitinated proteins. It should be cautious to interpret the result if the protein is overexpressed in cells.

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