Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Summary

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Abstract

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.

Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.

The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Introduction

In most eukaryotes, spindle microtubules must attach to a single region of each chromosome, termed as centromere. The kinetochore is a complex of proteins that are located at the centromere. Studying the timing of centromere and kinetochore protein’s movements and the structure of kinetochores and centromeres is important for understanding chromosome instability (CIN) and cancer progression. The key questions are how the chromosomal location and function of a centromere (i.e., centromere identity) are determined and how they participate in accurate chromosome segregation. In most species, the presence of a special nucleosome containing a specific histone-like protein called CENP-A defines the centromere identity. Therefore, it is proposed that CENP-A is the non-DNA indicator (epigenetic mark) of centromere identity. It is important to elucidate the mechanism of how CENP-A defines the centromere identity in humans.

The Holliday junction recognition protein (HJURP) is the CENP-A-specific chaperon which deposits CENP-A in centromeric nucleosomes^1,2,3. We have previously reported that the CUL4A-RBX1-COPS8 E3 ligase is required for CENP-A ubiquitylation on lysine 124 (K124) and centromere localization⁴. Also, our results showed that the centromere recruitment of newly synthesized CENP-A requires pre-existing ubiquitylated CENP-A⁵. Thus, a model was provided suggesting that CENP-A ubiquitylation is inherited through dimerization between cell divisions.

In contrast to our findings and those of Yu et al., negative results regarding the CENP-A and its centromeric localization were recently published⁶. The article claimed that CENP-A modifications on lysine 124 (K124) are dispensable for the establishment, maintenance, and long-term function of human centromeres, based on their negative results showing that the mutation of K124R did not affect CENP-A centromere localization neither cell viability⁶. However, there is enough room for debate in their results and conclusions, and we have already described what problem there could be in their previous publication⁷. Attention should be paid that they fused proteins with CENP-A, which have much larger molecular weights than the size of endogenous CENP-A: e.g., they fused ~30 kDa enhanced yellow fluorescent protein (EYFP) to ~16 kDa CENP-A and analyzed EYFP-CENP-A K124R fusion protein in their RPE-1 CENP-A^-/F knockout system. K124 ubiquitin is not expected to bind directly to HJURP based on structural predictions⁴, however, addition of mono-ubiquitin is predicted to have an impact on protein conformation of CENP-A. The protein of CENP-A conformation can be changed by the presence of a large fusion protein, and this conformational change may mask the structural changes caused by the loss of ubiquitylation. We suggest that the fusion of large-sized protein induces ubiquitylation at a lysine other than K124 in EYFP-CENP-A K124R mutant and this ubiquitylation at another site inhibits/masks the original K124R single mutant phenotype. Evidence that ubiquitylation occurs at different lysine in the CENP-A K124R mutant protein with a large tag protein (EYFP) was reported in our previous publication⁸. It was found that EYFP tagging induces ubiquitination of another lysine site of EYFP-CENP-A K124R and that EYFP-CENP-A K124R mutant binds to HJURP. As a result, this ubiquitylation at another site inhibits/masks the original K124R single mutant phenotype, and both EYFP-CENP-A WT and K124R mutants showed centromere localization (we used and compared pBabe-EYFP-CENP-A WT and K124R mutant, together with pBabe-EYFP control.). The results demonstrated that Flag-tagged or untagged CENP-A K124R mutants are lethal but can be rescued by a monoubiquitin fusion, suggesting that CENP-A ubiquitylation is indispensable to cell viability.

In recent years, many studies have developed different assays to identify posttranslational modifications (PTMs) of CENP-A protein and other centromere-kinetochore proteins both in vivo and in vitro^9,10,11. Analogous to the PTMs of histone proteins that are a major mechanism regulating the function of chromatin, PTMs of centromeric chromatin components are also involved in an essential mechanism to regulate the overall structure and function of centromeres. The majority of CENP-A PTM sites are specific to CENP-A-containing nucleosomes, although a few of them are conserved in histone H3, suggesting that modification of these residues contribute to the centromere-specific function. PTMs of CENP-A including phosphorylation, acetylation, methylation, and ubiquitylation were previously reported⁹, suggesting that CENP-A is subjected to a variety of PTMs and their combinatorial arrays on its amino terminus and C-terminus histone-fold domain. The importance of CENP-A modifications in multiple functions was revealed by many groups including ours. These functions involve CENP-A deposition at centromeres, protein stability, and recruitment of the CCAN (constitutive centromere-associated network)⁹. However, limited studies and findings of CENP-A PTMs are preformed where comparisons are made with one of canonical histones that directly or indirectly regulate their function. Technical reports focusing on the methodology to identify these CENP-A PTMs are also limited.

Because CENP-A ubiquitylation is required for CENP-A deposition at the centromere¹², inherited through dimerization between cell division⁵, and indispensable to cell viability⁸, the method to identify CENP-A ubiquitylation would be essential in future to study the functional activity, positioning, and structure of the centromere. Therefore, here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that the EYFP tagging induces ubiquitylation at a different lysine in the CENP-A K124R mutant protein⁸. Protocols of other control assays and analyses (immunofluorescence analysis, colony outgrowth assay, and in vivo ubiquitylation assay) are also presented to discuss the outcome of major mass spectrometry analysis properly.

Protocol

1. Cell culture and retrovirus transfection of pBabe-EYFP-CENP-A constructs

NOTE: EYFP-CENP-A is expressed from pBabe-EYFP-CENP-A at a similar protein level to endogenous CENP-A. Total cellular CENP-A protein is replaced with this EYFP-CENP-A after the disruption of the CENP-A^-/F allele by Cre recombinase as in RPE-1 CENP-A-/- cells⁶.

1. Preparation of the supernatant containing retrovirus using 293T packaging cells.
   1. Day 0: Spread 293T packaging cells on the 6-well culture plate (1.0 x 10⁶ cells/well). Culture cells in high-glucose DMEM with 10% FBS and 1% penicillin-streptomycin. Incubate the cells at 37 °C in an atmosphere of 5% CO₂ for 24 h.
      NOTE: For optimal results, empirically determine the cell density to use in seeding.
   2. Day 1: Prepare transfection reaction around 23 h after spreading (24 h point is 0 h point of the transfection).Transfect expression plasmid of each pBabe-EYFP (B3182), pBabe-EYFP-CENP-A WT (B3161), and pBabe-EYFP-CENP-A K124R (B3164) (see Table 1).
   3. Choose one combination of helper/packaging plasmids listed in Table 2 and add them to tubes containing one of the plasmids listed above. There are mostly these 3 combinations for helper/packaging plasmids (Table 2). Any combination worked in these experiments and showed similar transfection efficiency.
   4. Prepare 50 μL of reduced serum medium and mix 2.0 μg of each pBabe-EYFP (B3182), pBabe-EYFP-CENP-A WT (B3161), and pBabe-EYFP-CENP-A K124R (B3164) (see Table 1) adding one combination of helper/packaging plasmids listed in Table 2 (see below). Incubate this mixture at room temperature for 5 min. This mixture is solution A.
   5. Prepare another 50 μL of reduced serum medium and mix 1.5 μL of transfection reagents I and II, respectively (Table of Materials). Incubate this mixture at room temperature for 5 min. This mixture is solution B.
      NOTE: Optionally, add only 6.0 μL of transfection reagent III (polyethyleneimine [PEI]; 1.0 mg/mL) in solution B or add 6.0 μL of transfection reagent III in addition to transfection reagents I and II in solution B (Table of Materials).
   6. Mix solutions A and B together, and incubate at room temperature for 15 min.
   7. After washing the cultured cells once with PBS, add the mixture of solutions A and B (i.e., DNA-lipid complex) directly to each well of the 6-well culture plate that has 500 μL reduced serum medium. The final concentration of the plasmid is 3.3 μg/mL.
   8. After incubating the cells at 37 °C in an atmosphere of 5% CO₂ for 4.5 h, change the medium to high-glucose DMEM with 10% FBS and 1% penicillin-streptomycin. Put 2 mL/well of culture medium after the medium change in the 6-well culture plate.
   9. Culture the cells at 37 °C in an atmosphere of 5% CO₂ for 48 h after transfection. Perform retrovirus infection on Day 3 using the supernatant containing retrovirus.
2. Retrovirus infection of CENP-A^-/F RPE-1 target cells.
   1. Day 2: A day before infection, spread CENP-A^-/F RPE-1 target cells to transfect in the 6-well culture well (2.25 x 10⁵ cells/well). Culture cells in DMEM: F12 medium (Table of Materials) with 10% FBS and 1% penicillin-streptomycin.
      NOTE: Spread cells for colony outgrowth assays, immunofluorescence, and western blot analysis as control. For optimal results, empirically determine the cell density to use in seeding.
   2. Day 3 (infection day of the virus): At 48 h after transfection of 293T packaging cells, collect the supernatant containing virus and filter through 0.45 μm filter (do not use a 0.2 μm filter that will shear virus envelope). It is recommended to use polyethersulfone (PES) filters.
   3. Infect CENP-A^-/F RPE-1 cells with the virus. For one well of the 6-well culture plate, add 1 mL fresh media, 1 mL virus supernatant and polybrene to a final concentration of 8 μg/mL. Incubate CENP-A^-/F RPE-1 cells at 37 °C in an atmosphere of 5% CO₂ for 4 days until Day 7 after transfection.
      NOTE: The incubation period for CENP-A^-/F RPE-1 cell growth must be determined empirically by following analyses for optimal results.

2. Immunofluorescence analysis of cells containing pBabe-EYFP-CENP-A

1. Day 7: 4 days after retrovirus infection of pBabe-EYFP-CENP-A constructs, remove the culture medium by aspiration. Rinse cells once with TBS (25 mM Tris-HCl, pH 7.5; 125 mM NaCl). Apply TBS to the side of the culture wells to avoid disturbing the surface of cells.
   NOTE: The optimal time point for cell fixation must be determined empirically. Immunofluorescence signals of both EYFP-CENP-A WT and K124R are detectable at the centromere at least 4 days after retrovirus infection of pBabe-EYFP-CENP-A constructs even without Cre infection (data not shown, Figure 1C-E for the data with Cre infection).
2. Perform methanol cell fixation and immunofluorescence staining as described previously¹³.
3. As primary antibodies use an anti-GFP antibody (1:1,000 dilution) and an anti–CENP-B antibody (dilution ratio of 1:200) for a centromere location marker in TBS containing 4% goat serum (see Table of Materials for antibodies used).
4. Remove excess mounting medium with a paper towel and seal the edges of the coverslip with nail polish at the last step.
5. Refer to the previously described method¹³ for immunofluorescence image observation, acquisition, quantification, and analysis of remaining signals of EYFP-CENP-A at the centromere.
6. Perform image acquisition, processing including deconvolution, quantification, and analysis using Softwares A or Softwares B1 and B2 (see Table of Materials). Optionally use Softwares C1-C3 (see Table of Materials) for a confocal laser scanning microscope.
   NOTE: See 2.6.1 in Supplemental coding files for all commands used in Software A. See 2.6.2 in Supplemental coding files for all commands used in Softwares B1 and B2.

3. Colony outgrowth assays using pBabe-EYFP-CENP-A after retro-Cre virus infection

NOTE: The reason for performing this assay is to compare the cell viability between EYFP-CENP-A WT and K124R mutant after the disruption of the CENP-A^-/F allele by Cre recombinase (after the replacement of total cellular CENP-A protein).

1. Retrovirus transfection of pBabe-EYFP-CENP-A constructs.
   1. Perform retrovirus transfection of CENP-A^-/F RPE-1 cells as described in section 1. Culture cells in DMEM: F12 medium with 10% FBS and 1% penicillin-streptomycin for 72 h after the virus infection.
   2. Three days after retrovirus infection of pBabe-EYFP-CENP-A constructs (on Day 6), add blasticidin S (10 μg/mL) in wells containing transfected cells to be used for colony outgrowth assay and control experiments. Cells are grown at least 14 days after virus infection in the presence of blasticidin S. Change the medium containing blasticidin S every 5 days.
      NOTE: If cells reach about 80% confluence before seeding for the colony assay (3.2.7. and 3.2.8), passage the cells at 1:2 and 1:5 ratio by trypsinization and plating in a 6 well culture plate.
   3. Collect cells for western blot on Day 7 to confirm the protein expression of pBabe-EYFP-CENP-A constructs without Cre infection. Perform western blot analysis as described in section 4. The results are shown in Figure 1B (lanes 1-4).
   4. For colony outgrowth assay with Cre virus infection, keep the cells growing for 14 days after the virus infection in the presence of blasticidin S (i.e., grow cells in blasticidin S containing medium until Day 17 for the results presented here).
2. Retro-Cre virus infection of pBabe-puro-Cre
   NOTE: Day 0 in step 3.2.1 corresponds to Day 13 of in section 3.1.
   1. Day 0 to Day 3: Perform retrovirus transfection of CENP-A^-/F RPE-1 cells using pBabe-puro-Cre (B3027) as expression plasmid (see section 1 for details). Ensure that blasticidin S (10 μg/mL) is added in the culture medium.
   2. Day 4: Trypsinize cells to detach it from the plate. Plate 500 or 5,000 cells in triplicate in the 6-well culture plate. Culture cells in DMEM: F12 medium with 10% FBS and 1% penicillin-streptomycin.
   3. Day 5: Add blasticidin S (10 μg/mL) to the culture medium. For 5,000 or 500 cells’ plating, select cells with blasticidin S (10 μg/ml) 3-24 days (until Day 14 in [3.2.7]) or 3-28 days (until Day 18 in [3.2.8]) after virus infection of constructs (pBabe-EYFP-CENP-A constructs).
      NOTE: In this colony outgrowth assay, the transfection of pBabe-puro-Cre is added along with the transfection of of pBabe-EYFP-CENP-A. The transfection of pBabe-EYFP-CENP-A is performed in 3.1. The transfection of pBabe-puro-Cre is performed in 3.2.1.
   4. Day 10 (7 days after retro-Cre virus infection): Collect cells for western blotting analysis to confirm protein depletion of endogenous CENP-A after retro-Cre virus infection and/or protein expression of pBabe-EYFP-CENP-A constructs on the same Day 10.
   5. Perform western blotting as described in section 4 on the same Day 10. Results are shown in Figure 1B (lanes 5-7).
   6. Perform immunofluorescence analysis (section 2 above) to confirm that both EYFP-CENP-A WT and K124R localized to the centromere 7 days after inactivation of the remaining endogenous CENP-A allele. The results are shown in Figure 1C-1E.
   7. Day 14: Perform the colony outgrowth assay with the plate seeded with 5,000 cells. Fix cells for 10 min in methanol and stain for 10 min in a crystal violet solution (2.3% crystal violet, 0.1% ammonium oxalate, 20% ethanol (see Figure 2B). Count the number of colonies using the OpenCFU software (see Figure 2C).
   8. Day 18: Use the plate seeded with 500 cells. Fix and stain cells as described in step 3.2.7 (see Figure 2B). Count the number of colonies (see Figure 2C).

4. Western blot analysis using pBabe-EYFP-CENP-A

NOTE: Refer to the previously described method¹³ for Western blot analysis using antibodies indicated in Figure 1B and Figure 2A and Table of Materials for EYFP-CENP-A proteins.

1. Isolate proteins by lysing the cells grown with different virus infection. Then, use 20 µg of protein in one well of SDS PAGE gel. Transfer the proteins to a PVDF membrane as described previously¹³.
2. Wash the membrane 3x after incubations with primary-secondary antibodies. Detect and analyze the protein bands on the membrane with the infrared imaging system and/or the chemiluminescence imager for immunoblot detection. These results are shown in Figure 1B and Figure 2A.
   1. For the infrared imaging system to detect and analyze the protein bands, see step 4.2.1 in Supplemental coding files.
   2. Use the chemiluminescence imager to detect and analyze the protein bands, see step 4.2.2 in Supplemental coding files. For this system, use an ultra-sensitive enhanced chemiluminescent (ECL) substrate (Table of Materials).
   3. Optionally, use western blot stripping buffer to strip out pre-incubated antibodies from the PVDF membrane and reblot it with different antibodies for the next turn of western analysis. Empirically determine the optimized incubation time and temperature to use.

5. Cell Culture, transfection, and in vivo ubiquitylation assays using pQCXIP-EYFP-CENP-A

NOTE: The protein level of EYFP-CENP-A expressed from pQCXIP-vector is ~ 10x higher than the endogenous CENP-A protein level. The usage of this vector facilitates immunoprecipitation of a higher amount of the EYFP-CENP-A proteins, observation of the ubiquitylation bands of EYFP-CENP-A, and identification of the ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein through mass spectrometry analysis.

1. Transfection for in vivo ubiquitylation assays using pQCXIP-EYFP-CENP-A.
   1. Seed 36.2 x 10⁵ CENP-A^-/F RPE-1 cells in a 10 cm tissue culture dish. Culture cells in high-glucose DMEM with 10% FBS and 1% penicillin-streptomycin.
      NOTE: For optimal results, empirically determine the cell density to be used in seeding. Prepare at least 2 dishes for one immunoprecipitation (IP) sample to obtain a minimum of 1 mg total protein.
   2. Incubate the cells at 37 °C in an atmosphere of 5% CO₂ for 18 h.
   3. At 17 h after seeding, prepare the transfection reagents.
   4. Make solution A by mixing 6.7 μg plasmid of each pQCXIP-EYFP (B3252), pQCXIP-EYFP-CENP-A WT (B3254), pQCXIP-EYFP-CENP-A K124R (B3256) (Table 1) in 335 μL of reduced serum medium, and incubate at room temperature for 5 min. Add 6.7 μg plasmid of pCGN-HA-Ubiquitin (B2806) to all samples.
      NOTE: All the vectors are listed in Table 1.
   5. Make solution B by mixing 10.1 μL of transfection reagents I and II, respectively in 335 μL reduced serum medium, and incubate at room temperature for 5 min.
      NOTE: An optional step is to add only 40.2 μL transfection reagent III (polyethyleneimine [PEI]; 1.0 mg/mL) in solution B or add 40.2 μL transfection reagent III in addition to transfection reagents I and II in solution B (Table of Materials).
   6. Mix solutions A and B together, and incubate at room temperature for 15 min.
   7. After washing the cultured cells once with PBS, add the mixture of solutions A and B (i.e., DNA-lipid complex) directly to each of the individual 10 cm tissue culture dish that has 3.35 mL μL reduced serum medium.
      NOTE: The final concentration is 1.67 μg/mL of plasmid.
   8. Incubate the cells at 37 °C incubator with 5% CO₂ for 4.5 h. After 4.5 h, change the medium to high-glucose DMEM with 10% FBS and 1% penicillin-streptomycin.
   9. Culture the cells at 37 °C with 5% CO₂ for 48 h after transfection. Collect cells for cell lysates preparation.
2. Preparation of protein A beads bound with anti-GFP antibody.
   1. Take 25 μL (50% v/v) of protein A beads for one reaction of immunoprecipitation (IP). Wash with buffer A1 (Table of Materials) at least 3x to remove EtOH, and make 50% solution with buffer A1 (20 mM Tris-HCl, pH 7.4; 50 mM NaCl; 0.5% Nonidet P-40; 0.5% deoxycholate; 0.5% SDS; 1 mM EDTA; complete EDTA-free protease inhibitor reagent).
   2. Add 2.0 μL of anti-GFP antibody (Anti#76: Homemade antibody) to the beads prepared above and add 10x volume of buffer A1 comparing with net beads volume.
   3. Perform end-to-end rotation at 4 °C for 4-18 h. The optimal time length for end-to-end rotation must be determined empirically based on the efficiency of the immunoprecipitation.
   4. Centrifuge the beads at 100 x g for 1 min and remove the unbound supernatant. Add buffer A1 to re-make a 50% (v/v) solution of the beads. Use 25 μL (50% v/v) of this solution for one reaction of IP.
3. Immunoprecipitation (IP) using protein A sepharose beads bound with anti-GFP antibody.
   1. Lyse cells obtained in step 5.1.9 in buffer A1 by sonication and freeze-thaw process.
   2. Measure protein concentrations and normalize protein amounts among different IP samples. Remove 5% of the sample from each tube to run as 5% Input sample in SDS-PAGE.
      NOTE: The 5% Input sample can be frozen in liquid nitrogen and stocked at -80 °C, if it is not loaded within a day.
   3. Mix the rest of 95% lysate with 25 μL (50% v/v) of protein A beads bound to anti-GFP antibody that was prepared in step 5.2. Perform end-to-end rotation at 4 °C for 4-18 h.
      NOTE: The optimal time length for end-to-end rotation must be determined empirically.
   4. Centrifuge protein A beads with the protein bound to it (i.e, immunoprecipitates) with 100 x g for 1 min, and remove the supernatant. Wash the immunoprecipitates with buffer A1 by centrifuging at 100 x g for 1 min. Perform this step 4x.
   5. Mix the 5% Input and the rest of 95% immunoprecipitates with 2x and 4x SDS-PAGE loading buffer¹⁴, respectively. Boil these two samples for 5 min and then load them on a 10.0% denaturing SDS-polyacrylamide gel for electrophoresis in different lanes. Use bigger SDS-PAGE gel (e.g., 17 cm x 15 cm) for electrophoresis. If samples are run in smaller gel, it may not be possible to observe clear/sharp ubiquitylation bands.
   6. Perform western blot analysis as described in section 4 using the antibodies indicated in the previous report⁸. The result is shown in Figure 2A.
   7. Use western blot stripping buffer to strip out the pre-incubated antibodies from the PVDF membrane and reblot it with different antibodies for the next round of western blot analysis.

6. Mass spectrometry to identify the ubiquitylation site of the EYFP-CENP-A K124R mutant

1. Transfection for mass spectrometry analysis using pQCXIP-EYFP-CENP-A.
   1. Seed CENP-A^-/F RPE-1 cells in a 10 cm tissue culture dish. Check that cell density is 36.2 x 10⁵ cells per dish. Culture cells in high-glucose DMEM with 10% FBS and 1% penicillin-streptomycin. Prepare at least 20 dishes for one immunoprecipitation (IP) sample, to obtain at least 10 mg total protein (see 5.3).
      NOTE: For optimal results, empirically determine the cell density to use in seeding.
   2. Incubate the cells at 37 °C in an atmosphere of 5% CO₂ for 18 h. Prepare the transfection reagents ~ 23 h after spreading (24 h point is 0 h point of the transfection).
   3. At 17 h after seeding, prepare the transfection reagents and transfect cells as (4.1.3). Transfected cells have pCGN-HA-Ubiquitin (B2806) and pQCXIP-EYFP-CENP-A K124R (B3256).
   4. Incubate the cells at 37 °C in an atmosphere of 5% CO₂ for 48 h after transfection. Collect cells for cell lysates.
   5. Lyse cells in buffer A1 and perform immunoprecipitation as (5.2) and (5.3). Run these two of immunoprecipitation lysates as following (6.1.6) and (6.1.7).
      NOTE: In (6.1.6), run the sample in standard Tris-glycine gels. In (6.1.7), run the sample in the commercially available 4%-12% Bis-Tris protein gels. See also Discussion.
   6. Keep one sample for 10% of total immunoprecipitants to confirm EYFP-CENP-A ubiquitylation and to precisely determine the position of ubiquitinated EYFP-CENP-A as described in section 5. Run this sample in standard Tris-glycine gels. SDS-PAGE and western blot were performed as (5.3).
   7. Keep another sample for 90% of total immunoprecipitants for mass spectrometry analysis. Run this sample within two wells of the commercially available 4%-12% Bis-Tris protein gels. Perform Coomassie blue staining using Coomassie blue solution. Excise and cut out the gel region of 50-70 kDa (putative mono-Ub-EYFP-CENP-A K124R region). Use this gel region for mass spectrometry analysis.
2. Mass spectrometry analysis using in-gel digestion.
   1. Dice each gel slice of interest into small pieces (1 mm²) and place it into 0.5 mL of protein low binding tubes.
   2. Wash with 100 μL 50% (v/v) acetonitrile in 25 mM NH₄HCO₃, vortex 10-15 min, spin down, discard the supernatant, repeat 3x.
   3. Concentrate the sample using benchtop vacuum concentrator for 30 min to dry the gel pieces.
   4. Add 10 μL of 10 ng/μL sequencing grade trypsin and let the gel pieces to rehydrate for 5 min.
   5. Add 25 mM NH₄HCO₃ just enough to cover the gel pieces, digest at 37 °C overnight.
   6. Transfer the digested supernatant into a clean 0.65 mL siliconized tube. Add 50% (v/v) acetonitrile/5% (v/v) formic acid (30 μL or enough to cover), vortex 10 min, spin and transfer into the same extraction tube. Repeat 3x.
   7. Add 10 μL acetonitrile to the gel pieces, vortex 5 min, and spin down. Transfer the supernatant to the same tube.
   8. Concentrate the samples using benchtop vacuum concentrator to 2 μL, add 8 μL 3% (v/v) acetonitrile /2% (v/v) formic acid to the sample, vortex for 15 min, and spin down at 16,000 x g for 30 min. Samples are ready for mass spectrometry analysis.
   9. Perform MS data acquisition with LC-MS/MS using a liquid chromatography system (Table of Materials) coupled with a mass spectrometry instrument (Table of Materials).
   10. Inject 8 μL of reconstituted sample onto a reverse phase liquid chromatography (RPLC) column.
   11. Separate the peptides with a 2-80% gradient of solvent B in 60 min. Ensure that the gradient consists of an increasing percentage of solvent B from 2% to 22% in 40 min, 22% to 35% solvent B in 12 min, then climbing to 80% solvent B in 4 min, and finally holding at 80% solvent B for the last 4 min. Set the flow rate constant at 300 nL/min.
       NOTE: Solvent A contains 0.1% formic acid and 2% acetonitrile, solvent B contains 0.1% formic acid and 98% acetonitrile. All concentrations are shown as volume/volume.
   12. Collect mass spectrometry data using data-dependent acquisition mode. Briefly, collect MS spectra in 350–1500 m/z for 250 ms. Select the Top 50 intense precursors with charge 2–5 for further fragmentation. Collect MS/MS spectra in 100–2000 m/z for 100 ms, Exclude precursor ions from reselection for 15 s.
   13. For database searching, open the commercial software (see Table of Materials) to analyze mass spectrometry data on the desktop.
   14. To make a new search, click the “LC” button on the top menu. Then click the “Add” button to upload the original MS raw data files.
   15. Select “Human Protein ID” in the “Paragon Method” as the database searching method. Search the original MS raw data files against the UniProt Homo Sapiens database (containing 160,566 sequences, http://www.uniprot.org/proteomes/UP611385640).
   16. Set searching parameters as the following: select trypsin as the digestion enzyme, allow up to 3 missing cleavages, 4 modifications and 2-5 charges per peptide. Set mass error up to 20 ppm for the first search, and 0.02 Da for fragmented ions. Specify false discovery rate (FDR) thresholds for protein, peptide, and modification sites less than 1%. Set all the other parameters in the software to default values (see Figure 3 for mass spectrometry analysis).
   17. Click the “Save as” button on the right of the top menu, select a folder for storing the searching results, enter the search name and click the “Save” button.
   18. Click the “Process” button on the right of the top menu to start the searching. After the search ends, data with the entered search name will be automatically stored in the folder selected. The data can be easily opened by Software F.
   19. To obtain MS/MS spectra of any specific peptide, double click the search results to open it by Software F. First, click the protein in the protein list on the top of the menu, then click the peptide on the middle of the menu. The MS/MS of this peptide is shown on the bottom of the menu.
   20. To export and save MS/MS spectra, right-click on the MS/MS spectra on the bottom of the menu, select copy and then paste to a suitable file such as PPT or doc. format.

Results

EYFP-CENP-A K124 mutant shows ubiquitylation, interaction with HJURP, and no defects in centromere localization neither cell lethality. Here the system reported by Fachinetti et al. (2017)⁶ was re-constituted: in diploid human (RPE-1) cells carrying one disrupted and one ‘‘floxed’’ CENP-A allele (CENP-A^-/F), EYFP-CENP-A was expressed from the pBabe-EYFP retrovirus vector. In this system, the expression of endogenous CENP-A from the CENP-A^...

Discussion

Here we described methods of mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that the EYFP tagging induces ubiquitylation at a different lysine in the CENP-A K124R mutant protein⁸. In our results, we successfully identified ubiquitylation on lysine 306 (K306) in EYFP-CENP-A K124R, that is corresponding to lysine 56 (K56) in CENP-A through mass spectrometry analysis. The mass spectrometry analysis described here is a mimic method as we previously identif...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equpiments/Tools
0.5 ml protein low binding tubes	Eppendorf	022431064	For mass spectrometry analysis
10cm cell culture dish	BIOFIL/JET, China	700224	10 cm tissue culture dish (Yohei lab, PN63)
6 Well Cell Culture Cluster	Fisher/Corning Incorporated	07-200-83	6-well culture plate
CentriVap	LABCONCO	-	Benchtop vacuum concentrator for vaccum dry peptides for mass spectrometry analysis
ChromXP C18CL, 120A, 15 cm x 75 μm	Eksigent Technologies	805-00120	Liquid chromatography (RPLC) column for mass spectrometry analysis
HCX PL APO 100x oil immersion lens	Leica	LEICA HCX PL APO NA 1.40 OIL PHE	100X Oil immersion lens
HCX PL APO 63x oil immersion lens	Leica	LEICA HCX PL APO NA 1.40 OIL PH 3 CS	63X Oil immersion lens
Immobilon-FL PVDF Transfer Membrane	EMD Millipore	IPVH00010	For western blot
Leica DM IRE2 motorized fluorescence microscope	Leica	-	motorized fluorescence microscope
Leica EL6000 compact light source	Leica		External light source for fluorescent excitation
Micro Cover glass (22 mm x 22 mm)	Surgipath	105	Cover glass (22 mm x 22 mm)
Model V16-2 polyacrylamide gel electrophoresis apparatus	Apogee Electrophoresis/CORE Life Sciences	31071010	Gel electrophoresis apparatus I to apply bigger SDS-PAGE gel
nanoLC.2D	Eksigent Technologies	-	liquid chromatography system for mass spectrometry analysis
NuPAGE 4%-12% Bis-Tris Protein Gels	Thermo Fisher	NP0335BOX	The commercially available 4%-12% Bis-Tris protein gels for mass spectrometry analysis
Olympus FLUOVIEW FV3000 confocal laser scanning microscope	Olympus	-	Confocal laser scanning microscope (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519)
ORCA-R2 Degital CCD camera	Hamamatsu	C10600-10B	CCD camera
PAP Pen	Binding Site	AD100.1	For a water repellant barrier in immunofluorescent staining
TISSUE CULTURE DISHES 10CM	VWR	25382-166	10 cm tissue culture dish
Vertical electrophoresis for gel running (big size)	Junyi, China	JY-SCZ6+	Gel electrophoresis apparatus II to apply bigger SDS-PAGE gel (Yohei lab, PE23)
VWR Micro Slides, Frosted	VWR International	48312-013	Micro slides
Primary antibodies
Anti-CENP-A antibody	Stressgen/Enzo Life Sciences	KAM-CC006	Mouse monoclonal antibody
Anti-CENP-B antibody	Novus Biologicals	H00001059-B01P	Mouse monoclonal antibody
anti-GAPDH	ABCAM	ab37168	Rabbit polyclonal antibody
anti-GAPDH	Invitrogen	PA1987	Rabbit polyclonal antibody
anti-GFP antibody	ANTI #76 (Homemade antibody)		Rabbit polyclonal antibody
anti-HA (3F10)	Roche	11815016001	Rat monoclonal antibody
anti-HJURP	Proteintech Group	15283–1-AP	Rabbit polyclonal antibody
anti-Ubiquitin	Bethyl Laboratories	A300-317A-1	Rabbit polyclonal antibody
Reagents
Bio-Rad Protein Assay	Bio-Rad	500-0006	Commercial protein assay reagent I for measurement of protein concentration (compatible with 0.1% SDS)
Branson SONIFIER 450			Sonicator
Branson Ultrasonics sonicator Microtip Step, Solid, Threaded 9.5 mm	VWR Scientific Products Inc.	33995-325	Disruptor horn for sonication
Branson Ultrasonics sonicator Microtip Tapered 6.5 mm	VWR Scientific Products Inc.	33996-185	Microtip for sonication
Buffer A1	-	-	20 mM Tris-HCl, pH 7.4; 50 mM NaCl; 0.5% Nonidet P-40; 0.5% deoxycholate; 0.5% SDS; 1 mM EDTA; complete EDTA-free protease inhibitor reagent
Complete EDTA-free protease inhibitor cocktail	Roche	11-873-580-001	Complete EDTA-free protease inhibitor reagent for buffer A1
Coomassie brilliant blue R-250	BBI Life Sciences	CAS 6104-59-2	Coomassie blue solution for mass spectrometry analysis
Crystal violet solution (2.3% crystal violet, 0.1% ammonium oxylate, 20% ethanol)	SigmaI-Aldrich	HT90132-1L	For colony staining
DAPI	SIGMA-SLDRICH	D9542	For nuclear staining
DMEM: F12 Medium	ATCC	30-2006	DMEM: F12 Medium
Fetal Bovine Serum, certified, heat inactivated, US origin	Life Technologies/Gibco	10082	FBS (fetal bovine serum)
High-glucose DMEM (Dulbecco’s modified Eagle’s medium)	Life Technologies/BioWhittaker	12-604	high-glucose DMEM
Lipofectamin 3000	Life Technologies/Invitrogen	L3000	Transfection reagent I for chemical transfection
Lipofectamin 3000, P3000 solution	Life Technologies/Invitrogen	L3000	Transfection reagent II for chemical transfection
Methanol	Fisher	A412-4	Fixation reagant
Non fat powdered milk (approved substitution for carnation powdered milk)	Fisher Scientific	NC9255871 (Reorder No. 190915; Lot# 90629)	Non-fat skim milk
Opti-MEM I	Life Technologies/Invitrogen	31985	Reduced serum media
p-phenylenediamine	SIGMA-SLDRICH	P6001	For mounting medium
Penicillin, Streptomycin; Liquid	Fisher/Gibco	15-140	Penicillin-streptomycin
Poly-L-Lysine SOLUTION	SIGMA-SLDRICH	P 8920	Poly-L-Lysine, 0.1% w/v, in water
Polyethyleneimine [PEI]; 1.0 mg/ml	Polysciences	23966–2	Transfection reagent III for chemical transfection
Protein A sepharose CL-4B beads	GE Healthcare/Amersham	17-0963-03	Protein A sepharose CL-4B beads for in vivo ubiquitylation assays using pQCXIP-EYFP-CENP-A
Restore Western Blot Stripping Buffer	Thermo Scientific	PI21059	Western Blot Stripping Buffer I
Sequencing grade trypsin	Promega	V5111	For mass spectrometry analysis
SuperSignal West Femto Maximum Sensitivity Substrate	Thermo	34095	Ultra-sensitive enhanced chemiluminescent (ECL) substrate
UltraPure Distilled Water	Life Technologies/Invitrogen/Gibco	10977	Sterile tissue culture grade water
Western Blot Stripping Buffer II ((50 mM Tris-HCl, pH 6.85; 2% SDS; 50 mM DTT; 100 mM 2-Mercaptoethanol)	-	-	Western Blot Stripping Buffer II
Secondary antibodies
Alexa Fluor 488 Goat Anti-Rabbit IgG	Life Technologies/Invitrogen	A11008	fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Alexa Fluor 594 Goat Anti-Mouse IgG	Life Technologies/Invitrogen	A11005	fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody)
Softwares
Acquisition FV31S-SW software	Olympus	-	Sofware C1 (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519)
Analysis FV31S-DT software	Olympus	-	Sofware C2 (https://www.olympus-lifescience.com.cn/en/support/ downloads/#!dlOpen=%23detail847250519)
cellSens Dimension software Ver. 1. 18	Olympus	-	Sofware C3 (https://www.olympus-lifescience.com.cn/en/ software/cellsens/)
Image Studio Analysis Software Ver 4.0	LI-COR Biosciences	-	Software D
Molecular Imager Versadoc MP4000 System	Bio-Rad	-	Chemiluminescence imager for immunoblot detection
Odyssey CLx Infrared imaging System	LI-COR Biosciences	-	Infrared imaging system for immunoblot detection
OpenCFU saftware	-	-	For colony counting (http://opencfu.sourceforge.net/)
Openlab version 5.5.2. Scientific Imaging Software	Improvision/PerkinElmer	-	Software A
ProteinPilot Software version 4.5	AB SCIEX	-	Software F for mass spectrometry analysis
Quantity One 1-D analysis software	Bio-Rad	-	Software E
TripleTOF 5600+ System	AB SCIEX	-	Mass spectrometry instrument
Volocity version 6.3 3D Image Analysis Software (Volocity Acquisition)	PerkinElmer	-	Software B1
Volocity version 6.3 3D Image Analysis Software (Volocity Quantification)	PerkinElmer	-	Software B2