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.
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.
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 nucleosomes1,2,3. 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 vitro9,10,11. 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.
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.
2. Immunofluorescence analysis of cells containing pBabe-EYFP-CENP-A
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).
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.
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.
6. Mass spectrometry to identify the ubiquitylation site of the EYFP-CENP-A K124R mutant
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-/F allele could be disrupted by Cre recombinase6. Gene constructs of EYFP- CENP-A WT or K124R mutant (Figure 1A), which rescues the loss of endogenous CENP-A, were stably expressed when retroviral integration was performed. The remaining expression of endogenous CENP-A from the CENP-A-/F allele was then disrupted by Cre recombinase. The expression of endogenous CENP-A was not detected 7 days after the induction of Cre recombinase (Figure 1B, lanes 5-7). Both EYFP-CENP-A WT and K124R proteins expression was found to be at a similar level to the initial endogenous CENP-A protein level (Figure 1B, lanes 3, 4, 6, and 7). Both EYFP-CENP-A WT and K124R mutants showed centromere localization at 7 days after the disruption of the remaining expression of endogenous CENP-A from the CENP-A-/F allele (Figure 1C-1E). Both EYFP-CENP-A WT and the K124R mutant showed ubiquitylation and interaction with HJURP unlike the case of Flag-tagged or untagged CENP-A WT and the K124R mutant (Figure 2A; data not shown for Flag-tagged or untagged CENP-A)8. The cell viability was also addressed by performing the colony outgrowth assay 14 days after the disruption of the remaining endogenous CENP-A allele (Figure 2B). Both EYFP-CENP-A WT and K124R mutants showed a similar number of ‘‘rescued’’ colonies 14 days after the disruption of the remaining endogenous CENP-A allele (Figure 2B and 2C). Thus, our results are in line with those reported by Fachinetti et al.6.
Ubiquitylation at lysine 306 (K306) in EYFP-CENP-A K124R was revealed by mass spectrometry analysis. It was found that both EYFP-CENP-A WT and the K124R mutant shows ubiquitylation and interaction with HJURP unlike the case of Flag-tagged or untagged CENP-A WT and the K124R mutant (Figure 2A; data not shown for Flag-tagged or untagged CENP-A)8. These results suggest that the fusion of EYFP protein induces ubiquitylation at a lysine other than K124 in EYFP-CENP-A K124R mutant, and this ubiquitylation at another site promote the interaction of EYFP-CENP-A K124R with HJURP. Ubiquitylation at lysine 306 (K306) in EYFP-CENP-A K124R was revealed in CENP-A-/F cells by IP-mass spectrometry analysis (Figure 3A and Figure 3B). The lysine 306 (K306) in EYFP-CENP-A K124R corresponds to lysine 56 (K56) in CENP-A. Taken together, our results suggest that the fusion of large-sized protein (e.g., EYFP-tagging) induces ubiquitylation at a lysine other than K124 in CENP-A, and this ubiquitylation at another site inhibits/masks the original K124R single mutant phenotype.
Figure 1: EYFP-CENP-A K124 mutant localizes at centromeres. (A) Representations of the different CENP-A protein constructs tagged with EYFP (enhanced yellow fluorescent protein) at the N terminus. Red letters mark the position of K124R amino acid substitution in the indicated construct (left). The results of the assays performed in this study are shown (right). (B) Western blot analysis did not show the presence of detectable endogenous CENP-A. Immunoblots analysis to check for the presence of expression of the indicated rescue constructs (ca. 45 kDa) before and after Cre infection. The protein expression of pBabe-EYFP-constructs was confirmed before Cre infection (lanes 1-4), as well as after Cre infection (lanes 5-6). The absence of the endogenous CENP-A protein (ca. 15 kDa) was confirmed in the CENP-A-/- cell lines collected at 7 days after Cre infection (lanes 5-6). GAPDH protein was used as a loading control. (C) EYFP-CENP-A K124 mutant localizes at centromeres. CENP-A-/F RPE-1 cells were cotransfected with indicated constructs, cultured 7 days after retro-Cre virus infection, and immunostained. Visualization of DAPI (blue), EYFP (green), and endogenous CENP-B (red), which served as a centromere location control. Scale bar, 10 μm. (D) The localization patterns shown in (C) summarized as histograms. More than 200 interphase cells with EYFP-positive signals were counted per experiment (n ≥ 3 experiments), and the mean percentages (±SD) are shown. ‘‘Others (Non-centromere)’’ depicts mostly damaged cells, dead cells, or cells with nucleolar localization in interphase, which were observed because of transfection or other treatments. No significant (n.s.) difference was observed in K124R comparing to WT (Student’s t-test). (E) EYFP-derived signals at centromeres shown in (C) were quantified. Signals were normalized to those of WT, and the mean percentages (±SEM) are shown. Please click here to view a larger version of this figure.
Figure 2: EYFP-CENP-A K124 mutant is ubiquitylated and interacts with HJURP, and cell viability was not affected by K124R mutation of EYFP-CENP-A. (A) The EYFP-CENP-A K124 mutant is ubiquitylated and interacts with HJURP. In vivo ubiquitylation assay. CENP-A-/F RPE-1 cells were transfected with the indicated constructs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) using anti-GFP rabbit polyclonal antibody were detected by western blot analysis using the indicated antibodies. Putative di-Ub-EYFP-CENP-A (**) and putative mono-Ub-EYFP-CENP-A (*) are indicated. This figure has been modified from Niikura et al.8. (B) Representative images from the colony outgrowth assay as shown in the scheme (top) of two different conditions ([1] and [2]) for the indicated transfectants of EYFP-CENP-A. (C) Histograms summarizing colony survival of the experiments in (B). The mean percentages (±SEM) of more than 3 independent experiments (n ≥ 3) are normalized with the percentage of surviving colonies in EYFP-CENP-A WT (EYFP-WT). ****p < 0.0001 and **p < 0.01 compared with EYFP control (Student’s t-test). No significant (n.s.) difference was observed in K124R comparing to WT (Student’s t-test). Please click here to view a larger version of this figure.
Figure 3: Fragments of ubiquitylated EYFP-CENP-A K124R peptides were detected in by mass spectrometry analysis. (A) Evidence of ubiquitylation at lysine 306 (K306) of EYFP-CENP-A K124R in RPE-1 CENP-A-/F cells. The lysine 306 (K306) of EYFP-CENP-A K124R corresponds to lysine 56 (K56) in CENP-A. The LQKLRGGSTHLLIR peptide (coverage 95.9%, confidence 87.8%) identified by collision-induced dissociation analysis is displayed. The m/z (Da) values of the b (green) and y (red) ions in the spectra of (A) detected during fragmentation are highlighted with green in the table of (B). The ubiquitylation of K306 is confirmed by the LRGG motif (incomplete cleavage) of the b-3 ion (m/z 753.4730) and y-8 ion (m/z 1350.8328). (B) The table highlights the m/z (Da) values of the b (green) and y (red) ions in the spectra of (A). LQK[Umc]STHLLIR in the table of (B) indicates the LQKLRGGSTHLLIR peptide shown in (A). These figures have been modified from Niikura et al.8. Please click here to view a larger version of this figure.
B number | Relevant characteristic(s) | Reference | Source |
B3027 | pBabe-puro-Cre | Niikura et al., 2019 | Dr. Amruta Ashtekar, Dr. Lawrence S. Kirschner |
B3182 | pBabe-EYFP | Niikura et al., 2019 | – |
B3161 | pBabe-EYFP-CENP-A WT | Niikura et al., 2019 | Dr. Daniele Fachinetti, Dr. Don W. Cleveland |
B3164 | pBabe-EYFP-CENP-A K124R | Niikura et al., 2019 | Dr. Daniele Fachinetti, Dr. Don W. Cleveland |
B3031 | psPAX2 | Niikura et al., 2019 | Dr. John Thompson, Dr. Gustavo W. Leone |
D3032 | pMD2.g | Niikura et al., 2019 | Dr. John Thompson, Dr. Gustavo W. Leone |
B3189 | pGP | Niikura et al., 2019 | Dr. Kenji Tago (Jichi Medical Univeristy, Japan) |
B3190 | pCI-VSVG | Niikura et al., 2019 | Dr. Kenji Tago (Jichi Medical Univeristy, Japan) |
B3001 | pPAM | Niikura et al., 2019 | – |
B3252 | pQCXIP-EYFP | Niikura et al., 2019 | – |
B3254 | pQCXIP-EYFP-CENP-A WT | Niikura et al., 2019 | – |
B3256 | pQCXIP-EYFP-CENP-A K124R | Niikura et al., 2019 | – |
B2806 | pCGN-HA-Ubiquitin | Niikura et al., 2019 | – |
Table 1: Plasmid vectors used in this study.
B number | Helper/packaging plasmid vectors | Combination 1 | Combination 2 | Combination 3 |
B3031 | psPAX2 (lentiviral gag/pol vector) | 2μg | ||
D3032 | pMD2.g (lentiviral env vector) | 2μg | ||
B3189 | pGP (retroviral gag/pol vector) | 2μg | ||
B3190 | pCI-VSVG (retroviral env vector) | 2μg | ||
B3001 | pPAM (amphotropic helper vector encoding retroviral gag-pol-env) | 2μg |
Table 2: Combinations of helper/packaging plasmid vectors used in (1.1.3): Retrovirus transfection of pBabe-EYFP-CENP-A constructs.
Supplemental Coding Files. Please click here to download these files.
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 identify the lysine 124 (K124) ubiquitylation site of CENP-A WT-Flag12. Therefore, this method can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. The mass spectrometry analysis based on LC-MS/MS is commonly accepted to identify potential posttranslational modifications (PTMs) of a wide spectrum of proteins. Our combinatory methods consisting of several assays/analyses (i.e., in vivo ubiquitylation assay, colony outgrowth assay, and mass spectrometry analysis) could be recommended for researchers who are interested in identifying functional roles of ubiquitylation(s) of their target protein(s).
However, this protocol does not cover the detection of ubiquitylated bands and/or identification of site-specific ubiquitylation(s) of these proteins in living cells or a specific single cell during the whole cell cycle. These years optogenetic approaches are developed dramatically and giving a high impact on quantitative studies of cell-signaling systems. Optogenetics has originally provided approaches that precisely activate or inhibit individual neurons using single-component, microbial opsin-based systems. Currently, protein activity with unprecedented spatiotemporal precision can be controlled by exploiting natural genetically encoded photoreceptors, and various genetically encoded tools allow light-control of many biological processes including protein phosphorylation15. Therefore, optogenetics is a promising system to investigate spatiotemporal protein kinase signaling at the cellular and the entire organism levels. The number of light-controlled protein kinases is rapidly expanding, although the current number is still limited. However, the development of light-controlled protein ubiquitylation is delayed, and high molecular weight tagging of photoreceptors may disrupt the ubiquitylation of both WT and mutant proteins and functionally alter the native protein function as aforestated. Therefore, the development of lower molecular weight tagging or probing technique is urgently required to visualize ubiquitylation and/or to investigate spatiotemporal protein ubiquitylation signaling at the living cellular and the entire organism levels.
In the present study, EYFP vector control is essential for control assays and analyses (immunofluorescence analysis, colony outgrowth assay, and in vivo ubiquitylation assay) to discuss the outcome of major mass spectrometry analysis properly. Non-specific interaction with EYFP-protein is often observed in the immunoprecipitation experiment, thus EYFP vector control is indispensable to evaluate the true interaction of EYFP-fused protein(s). Our mass spectrometry analysis using EYFP-CENP-A K124R expressed in the RPE-1 CENP-A-/F cells did not show ubiquitylation on lysine sites in the EYFP polypeptide sequence. However, in other unknown conditions, it could be expected that the lysine site in the EYFP polypeptide sequence is ubiquitylated in EYFP- and/or other tagged fusion protein of high molecular weight.
For colony outgrowth assays, it is recommended to collect cells for western blot analysis to confirm protein depletion of endogenous CENP-A after retro-Cre virus infection and protein expression of pBabe-EYFP-CENP-A constructs. In this way, we can judge if the colony outgrowth phenotype is truly due to the sole expression of exogenous CENP-A WT or mutants. For any western blot analysis, if stripping is performed to reblot the membrane with different antibodies for the next turn of western blot analysis, one should use the same quantitative detection system as a previous turn’s blot for the next turn’s blot with different antibodies. One should make sure that the bands in the previous turn’s blot are undetectable at the aimed region of the membrane in the next turn’s blot with different antibodies. Or use the same quantitative detection system as previous turn’s blot and check if the incubation with the mere secondary antibody used in the previous turn’s blot does not lead to the detection of protein bands before starting the next turn’s blot with different antibodies. For in vivo ubiquitylation assay, it is important to run bigger SDS-PAGE gel using the apparatus to run bigger SDS-PAGE gel (Gel electrophoresis apparatus I or II). Empirically, bigger SDS-PAGE gel would separate protein bands clearly and enhance the sensitivity of the detection of the faint bands that could have been poorly detected in the smaller gel (i.e., protein bands appear sharper in bigger gel).
It is extremely important to choose proper SDS-PAGE gel to map post-translational modifications (PTMs) of a specific protein thorough LC-MS/MS. The most widely used gel system for SDS-PAGE is the Laemmli system, which uses Tris-glycine gels comprising a stacking gel component and the resolving gel component. In this classical system, the pH and ionic strength of the buffers used in the stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8) are different from the buffer used for running the gel (Tris, pH 8.3). Band distortion, loss of resolution, or artifact bands may be caused by the highly alkaline operating pH of the Laemmli system. The previous report described major causes of poor band resolution with the Laemmli system16, including instability and short expiration period of the resolving gel due to hydrolysis of polyacrylamide at the high pH, chemical alterations of sample proteins, reoxidation of reduced disulfides of cysteine residues of proteins, and cleavage of Asp-Pro bonds of proteins with heating at 95-100 °C in Laemmli sample buffer at pH 5.2. The commercially available 4%-12% Bis-Tris protein gels are Bis-Tris HCI–buffered (pH 6.4) and operated at pH ca. 7.0 unlike traditional Tris-glycine gels16. The numerous merits comparing with the Laemmli system are generated by the neutral operating pH of the Bis-Tris systems16, including high stability and long expiration period of the resolving gel, enhanced sample protein stability during electrophoresis at neutral pH leading to sharper band resolution and accurate results, and complete reduction of disulfides and absence of cleavage of Asp-Pro bonds. In this report, we could successfully map PTMs of EYFP-CENP-A K124R protein (Figure 3) using the commercially available 4%-12% Bis-Tris protein gels. Therefore, the commercially available 4%-12% Bis-Tris protein gels are highly recommended to use for this purpose.
For mapping post-translational modifications (PTMs) of a specific protein, in-gel digestion of target protein coupled with LC-MS/MS is widely used. In this study, we sought to identify ubiquitination sites EYFP-CENP-A K124R using affinity purification-mass spectrometry (AP-MS) strategy. Therefore, the first key step is to obtain a large amount of ubiquitinated EYFP-CENP-A K124R protein with high purity using immunoprecipitation from EYFP-CENP-A K124R overexpressing cells, which could greatly facilitate the identification and confirmation of ubiquitination sites of EYFP-CENP-A K124R by LC-MS/MS. To reduce the interference of non-ubiquitinated EYFP-CENP-A K124R protein, western blots of anti-GFP, anti-HA (Ub), anti-Ubiquitin (see section [6.1.6]), and Coomassie blue staining (see section [6.1.7]) were performed to precisely determine the position of ubiquitinated EYFP-CENP-A K124R on SDS-PAGE gel. Lastly, only a minimized area of gel band containing ubiquitinated EYFP-CENP-A K124R was excised for in-gel digestion coupled with LC-MS/MS to get optimized results. When the dried peptides are reconstituted before operating LC-MS/MS, 3% (v/v) acetonitrile /2% (v/v) formic acid was used to help better solubility and recovery rate of peptides. Sometimes the database searching could result in PTMs which does not truly exist due to the approximate assignment algorithm of search engine. Thus, another critical step is to confirm EYFP-CENP-A K124R ubiquitination sites via manually reviewing MS/MS of ubiquitinated peptides with help of Software F (Table of Materials), which could eliminate “unreal” ubiquitination sites introduced by approximate assignment by database searching. In summary, this AP-MS strategy has established an efficient and robust pipeline for the identification of EYFP-CENP-A K124R ubiquitination sites with crucial biological significance. More importantly, this pipeline could also be widely extended to investigate the PTMs of various functional proteins.
The authors have nothing to disclose.
We thank Chao-Jun Li at the Model Animal Research Center, Nanjing University for mass spectrometry analysis. We thank Yanmini Dalal, Tatsuo Fukagawa, and current researchers at the Model Animal Research Center, Nanjing University and Greehey Children’s Cancer Research Institute for their helpful discussion, experimental guidance, and reagents. We thank Don W. Cleveland, Daniele Fachinetti, Yanmini Dalal, Minh Bui, Gustavo W. Leone, John Thompson, Lawrence S. Kirschner, Amruta Ashtekar, Ben E. Black, Glennis A. Logsdon, Kenji Tago, and Dawn S. Chandler for their generous gifts of reagents. Y.N. was supported by Jiangsu Province ‘‘Double- First-Class’’ Construction Fund, Jiangsu Province Natural Science Fund (SBK2019021248), Jiangsu Province 16th Six Big Talent Peaks Fund (TD-SWYY-001), Jiangsu Province “Foreign Expert Hundred Talents Program” Fund (SBK2019010048), and National Natural Science Foundation in China (31970665). This study was partly supported by NCI grant R21 CA205659.
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 |