Plasmids, Cloning and Expression of HB-Ubiquitin, Cell Culture, and Transfections
The retroviral vector pQCXIP (BD Biosciences) was used to express yeast HB-ubiquitin (pQCXIP-HB-ubi). Viral particles were generated in 293 GP2 packaging cells and used to transduce HeLa cells according to standard protocols in order to establish a stable cell line expressing HB-ubiquitin. Stable cell lines were periodically maintained with puromycine for selection.
HeLa cells were cultured in DMEM supplemented with 10% FCS, 1 μM biotin and 1% antibiotic-antimycotic agent (Invitrogen) in 5% CO2 at 37 °C. All cell lines were tested for mycoplasma contaminations and periodically treated with plasminogen (InvivoGen, San Diego).
To differentially label MG132 treated and untreated cells expressing HB-ubiquitin, a SILAC DMEM medium was used (Thermo Scientific, Rockford, IL), lacking the two essential amino acids arginine and lysine. Heavy media were supplemented with 0.028 mg/mL 13C6 15N4 arginine (isotopic purity > 98 atom %) and 0.073 mg/mL 13C6 15N2 lysine (isotopic purity > 98 atom %) (Cambridge Isotope Labeling, Andover, MA) and the same amount of 12C14N-arg/lys was added to the light medium.
To inhibit proteasome activity, cells were treated with 10 μM MG132 (American Peptide, Sunnyvale, CA) for 90 min at 37 °C. The control cells were treated with the solvent (DMSO) in parallel.
Tandem Affinity Purification of Ubiquitinated Proteins from Cell Lysates
Cells were grown in 150 mm dishes (15 plates in experiment 1–3; 20 plates in the SILAC experiments 4 and 5). Cells attached to plates were washed twice with ice cold 1× PBS, pH 7.4, and harvested on plates with buffer A (8 M urea, 300 mM NaCl, 50 mM NaH2PO4, 0.5% NP-40), pH 8.0, and 1 mM PMSF.
Lysates were centrifuged at 15 000g
, 30 min, 20 °C, and the clarified supernatant was used for purification. 35 μ
L of Ni2+
sepharose beads (GE Healthcare) were used for each 1 mg of protein lysates and were incubated overnight at room temperature in buffer A with 10 mM imidazole on a rocking platform. Beads were pelleted by centrifugation at 100 × g for 1 min and washed sequentially with 20 bead volumes of buffer A (pH 8.0), buffer A (pH 6.3), and buffer A (pH 6.3) with 10 mM imidazole. After washing the beads, proteins were eluted twice with 5 bead volumes of buffer B (8 M Urea, 200 mM NaCl, 50 mM Na2
, 2% SDS, 10 mM EDTA, 100 mM Tris, 250 mM imidazole) pH 4.3. The pH of the elute was adjusted to pH 8.0. Ubiquitinated proteins were bound to 7 μ
L streptavidin sepharose beads (Thermo Scientific, Rockford, IL) for each 1 mg of initial protein lysate by incubation on a rocking platform overnight at room temperature. Streptavidin beads were washed sequentially with 2 × 25 bead volumes of buffer C (8 M Urea, 200 mM NaCl, 2% SDS, 100 mM Tris, pH 8.0) and buffer D (8 M Urea, 1.2 M NaCl, 0.2% SDS, 100 mM Tris, 10% EtOH, 10% Isopropanol, pH 8.0). After washing the beads 3 times with 25 mM NH4
, pH 8, the proteins were released by on-bead digestion with trypsin at 37 °C for 12–16 h on a rocking platform as described.5,14
Tryptic peptides were extracted 3 times using 25% (v/v) acetonitrile (ACN), 0.1% (v/v) formic acid (FA) and subsequently separated by strong cation exchange (SCX) chromatography, as previously described.15
Twelve fractions were manually collected, desalted, concentrated, and analyzed by LC–MS/MS as described.15
Liquid Chromatography, Tandem Mass Spectrometry, and Data Processing
LC–MS/MS was carried out by nanoflow reverse phase liquid chromatography (RPLC) (Eksigent, CA) coupled online to a Linear Ion Trap (LTQ)-Orbitrap XL mass spectrometer (Thermo-Electron Corp). Briefly, the LC separation was performed using a capillary column (100 μm ID × 150 mm long) packed with C18 resin (GL sciences) and the peptides were eluted using a linear gradient from 2 to 5% B over 5 min and 5 to 25% B over 90 min at a flow rate of 350 nL/min (solvent A: 100% H2O/0.1% formic acid; solvent B: 100% acetonitrile/0.1% formic acid). Nanoelectrospray was achieved using a pulled capillary tip with 10 μm ID (New Objectives, Woburn, MA) mounted on a packed tip stand manufactured by Thermoelectron Corp.; 1.7kV was applied on the tip. A cycle of one full FT scan mass spectrum (350–2000 m/z, resolution of 60 000 at m/z 400) was followed by 10 data-dependent MS/MS acquired in the linear ion trap with normalized collision energy (setting of 35%). Target ions already selected for MS/MS were dynamically excluded for 30 s.
Monoisotopic masses of parent ions and corresponding fragment ions, parent ion charge states and ion intensities from the tandem mass spectra (MS/MS) were obtained using an in-house software with Raw_Extract script from Xcalibur v2.4. Following automated data extraction, resultant peak lists for each LC–MS/MS experiment were submitted to the development version 5.0 of Protein Prospector (UCSF) for database searching similarly as described.16
A concatenated SwissProt (2007.11.07) database generated from the normal database and its reversed form (34,972 entries) was used for database searching. Trypsin was set as the enzyme with a maximum of two missed cleavage sites. The mass tolerance for parent ion was set as ± 20 ppm, whereas ±1 Da tolerance was chosen for the fragment ions. Following chemical modifications were selected as variable modifications during database search: protein N-terminal acetylation, methionine oxidation, N-terminal pyroglutamine, and deamidation of asparagine. Maximal modifications on a given peptide was set as 3. The Search Compare program in Protein Prospector was used for summarization, validation and comparison of results. To determine the expectation value cutoff that corresponds to a percent false positive (% FP) rate, the plot of the expectation values versus % FP rate for each search result was automatically obtained using the Search Compare Program. Based on these results, we chose an expectation value cutoff for all peptides corresponding to ≤1% FP. General protein identification is based on at least two peptides. For the SILAC samples, two additional variable modifications were included: 13C6 15N4-labeled arginine and 13C6 15N2-labeled lysine.
To quantify protein relative abundance changes, the Search Compare function was used to determine the light/heavy (L/H) ratios based on the intensities of the monoisotopic masses of the parent ion peptide pairs. Search Compare also corrects for the isotopic purity of the heavy SILAC amino acids, which was set to 98% purity with the signal/noise threshold set at 10. The peptide peak intensities were averaged across the elution profile (30 s) as described.16
For peptides matching to multiple members of a protein family, only the protein containing at least one unique peptide was reported. In the case that none of the proteins contain at least one unique peptide, all of the possibilities are reported with a “/” separating the protein names.
After protein identification, a second search was performed for each sample against the list of proteins identified with the given cutoff threshold to identified ubiquitination sites. During the second search, the following variable modifications were added: carbamylation of lysine, dioxidation of tryptophan, GlyGly modification of lysine, and phosphorylation of serine, threonine, or tyrosine.
All peptides with precise ubiquitination sites and ubiquitin chain linkage types were manually confirmed, considering the following 4 steps:
- Each MS/MS spectrum for validation was individually submitted for a database search, using the Swissprot concantenated database without species restriction.
- All of the b and y ion series were inspected manually to ensure the sensible interpretation based on peptide fragmentation rules (e.g., favorable cleavage at P site).17
- A series of consecutive y and/or b ions should be observed and all of the major ions have to be interpreted.
- MS/MS spectra of modified- and unmodified peptides have to be compared.
Cell Proliferation Assay
The growth rate of HeLa cells and HeLa cells expressing HB-tagged ubiquitin was determined by a colorimetric cytotoxicity assay, which measures the cellular protein content of cell cultures.18
In brief, cells were fixed with 0.4% trichloroacetic acid (wt/vol) and stained with sulforhodamine B dissolved in 1% acetic acid. The protein bound dye was extracted with 10 mM Tris base and the optical density was measured at 564 nm ().
Figure 1 HeLa cells stably expressing HB-tagged ubiquitin. (A) Schematic depiction of the HB-ubiquitin expression construct. The RGS6xHis epitope combined with a bacterially derived in vivo biotinylation signaling peptide was fused to ubiquitin. Expression was (more ...)
Proteins were separated by SDS-PAGE and transferred to PVDF membranes using a semidry blotting apparatus. Membranes were blocked in TBS containing 0.2% Tween-20 and 5% milk for 60 min and incubated in primary antibodies overnight at 4 °C. The RGS4His antibody (Qiagen, Valencia, CA) for detection of the RGS6xHis tag was diluted 1:2000 in TBS-Tween-20, 5% milk. HRP-conjugated secondary antibodies were used at a dilution of 1:15 000 in TBS-Tween-20, 5% milk. To detect the biotinylated portion of the HB-tag the membrane was incubated for 1–2 h at room temperature with HRP-conjugated streptavidin (1:10 000 in TBS-Tween) (Fisher, Pittsburgh, PA). Immunodetection was performed with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).