What's in Your Chain?

A poly-Ubiquitin chain that looks pure by SDS-PAGE and/or HPLC analysis may still contain significant amounts of a very troublesome contaminant: alternative linkages. The presence of unintended linkages in a poly-Ubiquitin chain can result in confusing results and erroneous conclusions. How would you know if the vial of K29 di-Ubiquitin you just purchased is devoid of other Ubiquitin linkages? By gel analysis most poly-Ubiquitin dimers look the same! Boston Biochem now gives you peace of mind by analyzing its poly-Ubiquitin products with a technique known as Absolute Quantitation of Ubiquitin (AQUA, Ub-AQUA, or AQUA-PRM). First developed by Donald Kirkpatrick et al. and subsequently refined by others, including Alban Ordureau et al., AQUA is now the Gold standard for precise, quantitative characterization of the various linkages present in a poly-Ubiquitin sample. In the AQUA method, all eight ubiquitinated “-GG” peptides are labeled with a stable isotope and then utilized as internal standards, which can be distinguished in a mass spectrometer (Figure 1). Boston Biochem is the only commercial source for AQUApure™ Ubiquitin chains that come complete with AQUA analysis results.

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Figure 1: Ub-AQUA overview

A: Tryptic digests of poly-Ubiquitin chains produce unique (“signature”) peptides that are specific to the presence of a particular linkage. For example, digests of K48-linked Ubiquitin contain a LIFAGKQLEDGR peptide with a -GG tag attached to the central lysine residue—other poly-Ubiquitin chains do not generate this particular species. Every poly-Ubiquitin chain type has a signature peptide that is specific to it—Ub-AQUA analysis takes advantage of this fact by using heavy-labeled versions of the signature peptides as internal standards (references) in an LC-MS analysis.

B: The Ub-AQUA workflow. Purified poly-Ubiquitin chains (or customer-supplied samples) are in-solution digested or isolated by SDS-PAGE and digested to generate tryptic peptides. Following clean-up, the digests are mixed with known quantities of isotopically-labeled AQUA control peptides. The mixtures are analyzed using an LC-MS/MS technique known as Parallel Reaction Monitoring, or PRM. AQUA-PRM allows the signals from sample peptides to be compared directly to the signals from the corresponding AQUA reference peptides. Using methods first developed by Donald Kirkpatrick et al. and subsequently refined by Alban Ordureau et al., a quantitative value for each type of poly-Ubiquitin chain linkage present in the sample is generated.