A number of large-scale analyses of ubiquitinated proteins were performed, and they are different from each other with respect to starting samples, purification schemes, and MS technologies. In S. cerevisiae, Peng and co-workers [17, 20] described an extensive study of identifying ubiquitinated proteins from a mutated strain, in which all native Ub genes were deleted in the genome, and only His-tagged Ub was expressed from an introduced plasmid. His-tagged Ub conjugates were purified by Ni affinity chromatography followed by in-solution digestion and LC/LC-MS/MS. The mock experiment was carried out with an isogenic yeast strain expressing wild-type Ub. After removing 48 proteins in the mock, more than 1000 proteins were determined by MS in the pre-enriched sample. In addition, 110 ubiquitinated sites were defined in the sequences of 72 identified proteins, confirming the modification on these proteins. However, the majority of -GG peptides were missed in the analysis, highlighting the undersampling of "shotgun" MS. Although the exact FP rate is not known, it is believed that most of the identified 1000 proteins are modified by Ub . Moreover, immunoprecipitation was used to assess the ubiquitination of 19 proteins, and none were found to be FP. Later, a TAP strategy under denaturing condition was developed to increase the purity of Ub conjugates, leading to identification of more than 250 proteins in yeast . The tandem tag is composed of six His residues and a biotin motif. The tag is efficiently biotinylated by endogenous biotin ligases in vivo, which exist in both prokaryotic and eukaryotic cells. The extremely strong binding of biotin to streptavidin resin allows very stringent wash under denaturing conditions. In addition, Mayor et al.  implemented an affinity purification step by a Ub receptor Rpn10 in yeast prior to denaturing Ni column. The method permits the analysis of some ubiquitinated substrates that are recognized by a specific Ub receptor, which has potential for investigating Ub receptor-substrate interactions in cells.
Similarly, the ubiquitinated proteins from mammalian cells were examined by several groups with some success. Kirkpatrick et al.  found ~20 proteins in Ub-conjugate enriched samples from cells transfected with His-tagged Ub. Some researchers tested affinity purification of native Ub conjugates using Ub antibodies [19, 24]. Matsumoto et al.  attempted both native and denaturing conditions for the antibody affinity capture, and identified 670 and 345 proteins, respectively, indicating that ~50% of proteins enriched under native conditions are associated with the columns despite that they are not modified by Ub. In addition, Gururaja et al.  used an in vitro ubiquitination assay to synthesize His-tagged Ub conjugates from HeLa cell extract for subsequent proteomics analysis. Taken together, the methodology in mammal at current stage is not as mature as that in yeast, because the mammalian cells contain much more native His-rich proteins for Ni affinity chromatography, and the expression level of tagged Ub is often lower than that of endogenous Ub in mammals. Further improvement of the method is required for routine analysis of proteins modified by Ub in more complex proteome. The tandem affinity tag  may be better suited for such a challenge.
The development of technology for profiling Ub conjugates makes it possible to compare ubiquitinated proteins under different physiopathological conditions. For instance, Hitchcock et al.  used the strategy to identify numerous membrane-associated Ub substrates in the ER-associated degradation (ERAD) pathway. In the experiments, they generated yeast strains with mutations in both the ER resident ubiquiti-nating enzyme UBC7 and the ERAD-associated protein NPL4. A total of 83 potential substrates in the ERAD pathway were found when compared to the wild-type strain.
Was this article helpful?
Not Able To Lead A Happy Life Because Of Excessive Allergies? Want To Badly Get Rid Of Your Allergy Problems, But Are Super Confused And Not Sure Where To Even Start? Don't Worry, Help Is Just Around The Corner Revealed The All-In-One Power Packed Manual Containing Ample Strategies And Little-Known Tips To Get Rid Of Any Allergy Problems That Are Ruining Your Life Learn How You Can Eliminate Allergies Completely Reclaim Your Life Once Again