Ubiquitin (Ub) is a highly conserved protein found throughout the eukaryotic kingdom.1,2
The covalent conjugation of Ub to protein substrates plays an essential role in various cellular processes.3–8
Ubiquitinated proteins can be either mono- or poly-ubiquitinated, or both, depending on their fate and function.7,9–11
Substrate–Ub conjugation occurs via
an amide bond between the ε-amino group of a lysine residue from the substrate and the carboxylic group of the C-terminal glycine (G76) of a Ub. Ubiquitin conjugation is catalyzed by ubiquitination enzymes (E1, E2, and E3) in a multi-step process.12–17
Poly-ubiquitination of a substrate happens when multiple Ub proteins are conjugated to a substrate-linked Ub via
an inter-ubiquitin linkage. Inter-ubiquitin conjugation is the result of the same chemical reaction that catalyzes the attachment of Ub to the substrate protein and is catalyzed by the same enzymes (E1, E2 and E3). The only difference is that the lysine involved in the amide bond formation is from a Ub instead of a substrate protein.18
There are seven lysine residues in the Ub sequence (K6, K11, K27, K29, K33, K48, and K63) and all are known to participate in the formation of poly-Ub chains. Poly-Ub chains are mostly formed via
K48 and K63 and poly-Ub chains formed via
other lysines constitute a small portion of all linkages.8
Poly-Ub chains can be formed linearly where only one lysine in each Ub is involved in linkage formation or branched where multiple lysines from a Ub chain are involved in linkage formation. Linkages in the poly-Ub chains can be via
the same lysines (homogeneous) in all chains or different lysines (heterogeneous). Even though the relationship between the type of linkages in a poly-Ub chain and their role in determining the modified protein’s fate is poorly understood, there are some hints that K63 is involved in stress response (non-degradative ubiquitination) while all other linkages play a role in protein degradation (degradative ubiquitination).8
The ability to analyze poly-Ub chains and their attachment sites to substrate proteins has significantly advanced in recent years. Large scale proteomic studies have been conducted successfully to identify ubiquitinated substrates and the respective Ub attachment sites.11,9–24
In most of these studies, the focus has been on identifying the ubiquitinated proteins and the site(s) of ubiquitination, while there has been less emphasis on determining the composition and topology of the poly-Ub chains. Determining the mono- or poly-ubiquitination status of the substrate as well as density (# of inter-ubiquitin linkages per unit of poly-Ub) and structure (type of inter-ubiquitin linkages) of the Ub moiety is as important as the identity of the substrate itself. That is because often the structure of the Ub moiety determines the substrate’s fate.8
Mono- and poly-ubiquitinated forms of a substrate can be distinguished by immunoblotting due to the significant contribution of the Ub moiety to the total mass of the protein conjugate. However, the linkages in poly-Ub chains cannot easily be characterized by this approach unless linkage-specific antibodies are used. The first limitation of characterizing poly-Ub chains by immunomethods relates to the development of suitable antibodies which so far have only been generated for K48 and K63 linkages.25
The second limitation relates to the binding mechanism of such antibodies which is more based on structural recognition rather than sequence recognition. This means that linkage specific antibodies recognize the linkages based on the structural features that they induce in poly-Ub chain topography, so the linkage can only be characterized on folded proteins. Even though these antibodies show a high level of affinity (Kd
(K48) = 1.2 nM, Kd
(K63) = 92 nM) and specificity for their targets, higher amounts of poly-ubiquitinated proteins are often required for detection because most proteins are fully or partially denatured during SDS-PAGE electrophoresis which eliminates structural epitopes. The third limitation arises from the fact that a significant fraction of ubiquitinated substrates exhibit heterogeneous poly-Ub chain linkages.25
The poly-Ub chain heterogeneity and lack of linkage specific antibodies for all linkages make the task of determining linkage frequency by immunoblotting even more complicated. The fourth limitation of linkage specific antibodies is in relative linkage quantification which requires comparing the western blot signals of different linkage specific antibodies.
Selected Reaction Monitoring (SRM), frequently referred to as multiple reaction monitoring (MRM), is a highly sensitive and quantitative mass spectrometric technique which is increasingly being used for proteomic applications. The method uses a series of quadrupole mass analyzers to detect and quantify specific, predetermined peptides in complex mixtures accurately and sensitively. SRM has been used before for the characterization of poly-Ub chain connectivity. Specifically, the Gygi group used 10 heavy isotope-labeled Ub peptides to develop an SRM assay that was used to characterize the ubiquitinated cyclin B1.26,27
However, a complete assay for comprehensive characterization and quantification of Ub and its linkages has not been described. Ubiquitination is a complex PTM with various manifestations and a systematic investigation of this PTM by SRM requires a comprehensive assay that targets all ubiquitin forms in their entirety. An ideal assay should include all peptides derived from ubiquitin to deal with issues such as: (1) variations in Ub peptide concentrations as a result of digestion inconstancies,26,28
(2) the fact that none of the tryptic peptides derived from a poly-Ub chain can be used individually to measure Ub concentration even when the digestion is complete and all peptides exist in equimolar ratios relative to intact Ub, and (3) PTMs of Ub itself. Previous studies have mainly focused on linked peptides derived from human poly-Ub digestion and a few unmodified peptides as mono-Ub representatives while neglecting the PTM-bearing Ub peptides altogether. In addition, previously targeted peptides have been assayed based on single transitions without extensive optimizations for maximum sensitivity.26
Such transitions may be sufficient for detection of peptides extracted from in-gel digestion of intact proteins but would certainly not be specific and sensitive enough for unambiguous detection of Ub peptides in complex mixtures. Previous assays have also not been well-characterized in terms of limit of detection (LOD) and linear dynamic range (LDR) which are necessary for high throughput analysis of substrates varying widely in concentration.
Here we introduce a complete set of SRM assays for the detection and quantification of ubiquitin and all inter-Ub linkages in yeast and human. Collectively, these assays represent a comprehensive, highly sensitive and specific method to probe the connectivity of poly-Ub chains. We show that the method is sensitive enough to measure the frequency of inter-ubiquitin linkages at the enriched protein level and specific enough to detect them in complex mixtures such as whole cell lysates. We used these assays to determine the frequency and density of different inter-ubiquitin linkages in synthetic human poly-Ub chains as well as in fifteen poly-ubiquitinated proteins enriched from yeast lysates to determine the level of degradative and non-degradative ubiquitination for each. We also used the assays to detect changes in the linkage density and frequency in an enriched sample of histone protein Htb2 and whole cell lysate when cells were treated with the DNA modifying agent methyl methanesulfonate (MMS). We demonstrate for the first time that Htb2 is poly-ubiquitinated. We then studied the effect of cadmium poisoning on Ub chain linkage density and frequency in a sample enriched for the transcription activator Met4, or whole cell lysate.