Ub-clipping: an approach for studying ubiquitin chain architecture

The post-translational modification of proteins with ubiquitin is a dynamic multifaceted process The complexity arises from their ability to form architecturally distinct polyubiquitin chains 1-3 . Despite our understanding of the importance of these signals, we currently lack tools and methods to study them. Here we describe an approach, termed Ub-clipping, which provides unprecedented insight into ubiquitin chain architecture. This protocol is related to our recent Nature paper titled, “Insights into ubiquitin chain architecture using Ub-clipping”. This technology takes advantage of an engineered viral protease, Lb pro *, which ‘clips’ ubiquitin such that the information on the site of modification is retained and the remaining ubiquitin and substrate polypeptides are kept intact. The goal of this protocol is to allow researchers to efficiently adapt our new technology to their proteomic workflows. We anticipate this method will continue to shed light on the architecture of ubiquitin signals, and therefore further our understanding of the ubiquitin code across a broad spectrum of biological systems.


Introduction
In this protocol, we introduce an engineered viral protease, Lb pro * from foot-and-mouth-disease virus, as a novel tool to study the complexity of ubiquitin modifications. Unlike canonical deubiquitinases which hydrolyze the isopeptide bond following the C-terminal GlyGly motif, Lb pro * specifically cleaves the peptide bond preceding the GlyGly motif. Consequently, this motif remains attached to the modification site. In our recent paper we show this unique proteolytic activity can be used to dissect · Extraction buffer: 4 M urea, 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 5% glycerol, 0.1% IGEPAL CA-  2. Sonicate the resuspended cells on ice (recommended sonication settings: 10 sec burst, 10 sec off, amplitude 60 W for 3 min).
3. Clear the cell lysate by centrifugation at 50,000 x g for 30 min at 4°C.

4.
Remove the supernatant and, together with a magnetic stir bar, place into a 100 mL beaker on ice overtop a stir plate at 4 °C.

(C) Purification
Ammonium sulfate precipitation 4. Analyze the ion-exchange run on an SDS-PAGE gel (Fig. 1b). Note: At this point in the purification it is expected to have contaminating proteins in the Lb pro *-containing fractions. However, these will be removed in subsequent steps of the purification.
7. Scrape off cells and transfer to a pre-chilled 1.5 mL tube.
8. Centrifuge at 300 x g for 2 min at 4 °C.
9. Remove and discard the supernatant.
10. Flash-freeze the cell pellet in liquid nitrogen or proceed with the protein extraction procedure described below. 10 μM Lb pro * is sufficient to collapse the ubiquitin smear (Fig. 2). For WT Lb pro , we recommend using a concentration higher than 10 μM during digestion.
2. Add Lb pro -treated sample to a pre-soaked dialysis tubing or cassette (3.5K MWCO).
3. Dialyze sample in cold Milli-Q water overnight at 4 °C with gentle stirring.
12. Incubate TUBE-bound ubiquitin chains with 100 µL of 10 µM Lb pro * in TUBE digestion buffer for 16 h at 37°C. Note: This is an on-bead cleavage assay, where Lb pro * clips and releases ubiquitin into supernatant.
the inhibitors.
14. Centrifuge at 10,000 x g for 10 min at 4 °C.
16. Resuspend the pellet in 1 mL of sucrose storage buffer.
17. Determine the protein concentration using a BCA assay. Note: Samples can be stored at -80 °C until needed. 11. Remove the supernatant and transfer to a fresh tube.
12. Protein can be immediately flash-frozen in liquid nitrogen or aliquoted and lyophilized. Store sample at -80 °C.

Mass spectrometry & Ub-clipping analysis
Mass spectrometry analysis will largely depend on the available equipment. As a result, in this section of the protocol we have minimized instrument-specific details and provided general guidelines for analysis and data processing of samples. For specific details on reagents and instrumentation, please see the method section of our paper.
(A) Mass spectrometry 1. Resuspend sample in reconstitution buffer. Note: The resuspension volume will depend on the sample and sensitivity of the mass spectrometer.
2. Desalt your sample using C 4 reverse phase media before mass spectrometry analysis. Note: This can be performed before or during HPLC runs.
3. Prior to ionization, separate ubiquitin species with an analytical column packed with C 4 reverse phase media.
4. Standard acetonitrile gradients can be used to elute ubiquitin from the analytical column.
5. We recommend performing mass analysis at the highest mass resolution setting possible.

(B) Data analysis
1. The analysis of ubiquitin modifications from mass spectrometry data files can be performed by peak integration or spectra deconvolution. Note: In many cases both approaches yield comparable results. However, spectra deconvolution is preferred when quantifying both phosphorylation and ubiquitination sites.
2. For peak integration, quantitation should be performed on the ubiquitin species with the highest intensity (e.g. ubiquitin with a charge state of 12) and the nominal mass. After peak integration, export the area under the curve for each species and quantify their relative abundance.
3. For spectra deconvolution, all spectra corresponding to ubiquitin should be averaged and deconvoluted. After spectra deconvolution, export the intensities of each ubiquitin species and quantify their relative abundance.
Troubleshooting Lb pro expression · If cells lyse during overnight expression or expression levels are unexpectedly low, try expressing Lb pro * for 5 h at 30 °C while shaking at 150 r.p.m.

Generation of substrates for Ub-clipping
· The generation of substrates for Ub-clipping analysis is a critical step in this protocol. In our experience, problems often arise due to a lack of initial starting material or inefficient isolation of ubiquitinated substrates. Therefore, as a first resort to troubleshooting this protocol, we often recommend increasing the amount of starting material and optimizing the conditions for substrate isolation.
· After Lb pro * cleavage, ubiquitin species can be purified in a number of different ways. In this protocol we described two different methods: 1) purification by size-exclusion chromatography and 2) perchloric acid extraction of ubiquitin. These methods are not mutually exclusive. For instance, the perchloric acid extraction method might be used to isolate total ubiquitin from whole cell lysates.
Therefore, if your laboratory is not equipped with an Äkta micro, we suggest performing perchloric acid extraction instead.

Mass spectrometry
· Troubleshooting mass spectrometry data acquisition/analysis parameters will largely be instrument specific, however routine maintenance of instrumentation often helps with reproducibility and sensitivity issues.

Ub-clipping analysis
· Small chemical post-translational modifications of ubiquitin (e.g. phosphorylation) will alter the charge state of the modified ubiquitin. This will bias the quantitation of ubiquitin species using peak integration, which relies on a uniform charge state. Therefore, in instances where ubiquitin phosphorylation is identified we recommend quantifying by spectra deconvolution.

Anticipated Results
This protocol will produce Lb pro *-generated mono-ubiquitin species from both simple and complex ubiquitin samples (e.g. whole cell lysates, purified ubiquitin chains, purified mitochondria, etc.), while preserving information on linkage-type, additional post-translational modifications, and combinatorial complex modifications, including branched chains. These ubiquitin species can subsequently be analyzed by quantitative mass spectrometry to determine the extent of each ubiquitin modification.
Ub-clipping, therefore, provides insight into the complexity of ubiquitin modifications and allows for the modelling of ubiquitin chain architectures. Figure 1 a, Elution profile of Lbpro* from Resource Q anion exchange. The absorbance was measured at 280 nm and the peak corresponding to Lbpro* is labelled. b, Coomassie-stained SDS-PAGE of samples from a. The highlighted Lbpro*-containing fractions were pooled and concentrated. c, Fractions as in b were pooled from six separate anion exchange runs and further purified by size exclusion chromatography. The absorbance was measured at 280 nm and the peak corresponding to Lbpro* is labelled. d, Coomassie-stained SDS-PAGE of samples from c.