Chromatin-associated nonhistone proteins (CHRAPs) are readily collected from the DNaseI digested crude chromatin preparation. In this study, we show that the absolute abundance-based label-free quantitative proteomic analysis fail to identify potential CHRAPs from the CHRAP-prep. This is because that the most-highly abundant cytoplasmic proteins such as ribosomal proteins are not effectively depleted in the CHRAP-prep. Ribosomal proteins remain the top-ranked abundant proteins in the CHRAP-prep. On the other hand, we show that relative abundance-based SILAC-mediated quantitative proteomic analysis is capable of discovering the potential CHRAPs in the CHRAP-prep when compared to the whole-cell-extract. Ribosomal proteins are depleted from the top SILAC ratio-ranked proteins. In contrast, nucleus-localized proteins or potential CHRAPs are enriched in the top SILAC-ranked proteins. Consistent with this, gene-ontology analysis indicates that CHRAP-associated functions such as transcription, regulation of chromatin structures, and DNA replication and repair are significantly overrepresented in the top SILAC-ranked proteins. Some of the novel CHRAPs are confirmed using the traditional method. Notably, phenotypic assessment reveals that the top SILAC-ranked proteins exhibit the high likelihood of requirement for growth fitness under DNA damage stress. Taken together, our results indicate that the SILAC-mediated proteomic approach is capable of determining CHRAPs without prior knowledge.
Chromatin is a complex of DNA and proteins, in which the histones H2A, H2B, H3, and H4 are the major protein constituents [
Fission yeast is a useful model for analysis of RNA interference (RNAi) directed heterochromatin formation [
A traditional assay for testing whether a protein of interest is associated with the chromatin includes the preparation of CHRAPs extracts (or CHRAP-prep) through collection of the released proteins from the DNaseI digested crude chromatin and western blot analysis [
We found that the top ranked proteins by levels of abundance in CHRAP-prep were predominated by the ribosomal proteins, suggesting that the highly abundant non-CHRAPs are not effectively removed in CHRAP-prep. Hence, simply based on the level of protein abundance in CHRAP-prep by using the high-throughput proteomic analysis is unlikely to reveal CHRAP candidates without prior knowledge. SILAC (stable isotope labeling with amino acids in cell culture)-mediated proteomic analysis has shown to permit the quantitative analysis of the relative protein levels between those labeled with and without heavy isotopes [
In the SILAC proteomic analysis, SILAC-labeled samples are required to be fully incorporated with the heavy stable isotope-coupled lysine (e.g., 13C6-lysine or heavy-lysine) or arginine (e.g., 13C6-arginine or heavy-arginine) or both. To avoid the arginine-conversion problem [
Heavy lysine is efficiently incorporated in fission yeast. (a) A schematic diagram shows the consecutive subculturing of cells in heavy lysine containing medium. (b) An MS spectrum of the Eno1 peptide AVGNVNNIIAPVVK. The spectrum of the peptide resulted from passages p0, p1, p2, and p3 is shown. Green and red lines indicate the
The relative level of light versus heavy peptides/proteins was exemplified by the Eno1 peptide AVGNVNNIIAPVVK in various subcultures. As expected, no Eno1 peptide was detected to contain heavy-lysine in the initial culture (p0) prior to subculturing with the heavy lysine-containing medium (Figure
To further test whether the heavy lysine was uniformly incorporated in all peptides/proteins besides Eno1, a slice of SDS-PAGE gel containing proteins derived from the first subculture was subjected to LC-MS/MS analysis. The ratio between light and heavy peptide levels in all of the ~100 peptides detected was found to be close to −3 in log 2 scale, indicating that the heavy lysine is uniformly incorporated in all proteins in the culture (Figure
Next, we wanted to assess the sensitivity by varying the ratios of light and heavy peptides/proteins in the given premixed samples. A slice of SDS-PAGE gel (i.e., containing >100 peptides) from each given sample was subjected to the proteomic analysis after in-gel trypsinization. To this end, the distribution of ratios of all peptides detected in SILAC analysis correlated well with the expected ratio in the given samples, suggesting that our SILAC protocol is adequate for the quantitative proteomic analysis (Figure
High sensitivity of peptide/protein ratio detection by SILAC analysis. (a) Distribution of detected ratios by SILAC is correlated with the expected ratio in the given samples.
It has been shown that a given proteins can be tested for its association with the chromatins by enriching the CHRAPs [
Abundant ribosomal proteins are effectively depleted in CHRAP-prep by SILAC. (a) A schematic diagram shows the steps of CHRAP preparation. WCE stands for whole cell extract; sup for supernatant; pel for pellet; and resusp. for resuspension. (b) The image of the agarose gel shows the presence or absence of genomic DNA (gDNA) in various samples indicated as in (a). (c) The image of western blot shows the presence or absence of histone H4 and soluble protein tubulin (tub). (d) Top 10% ranked proteins by SILAC ratios are depleted of ribosomal proteins.
We judged that the absolute abundance of the majority enriched CHRAPs might not be higher than that of the depleted ribosomal proteins due to their high abundance prior to the enrichment. On the other hand, CHRAPs would be top ranked by comparing levels of enrichment or ratios between CHRAP-prep (after enrichment) and WCE (before enrichment). To test this possibility, equal amounts of CHRAP-prep and heavy lysine-labeled WCE proteins were mixed and subjected to LC-MS/MS analysis using LTQ Orbitrap mass spectrometry (see Section
A total of 507 proteins were identified based on the presence of paired light (i.e., CHRAP-prep) and heavy (i.e., WCE) peptides after selection of high quality peptide through the Trans-Proteomic Pipeline (
We noted that the occurrence of ribosomal proteins in the second top 10% ranked proteins by SILAC ratios was not reduced when compared to the background level (i.e., 14% versus 9.5%). This result suggests that the enrichment for CHRAPs in the second top 10% ranked proteins was less effective. Hence, only the first top 10% ranked proteins by SILAC ratio were considered for enriching CHRAP candidates and were further analyzed in this study.
CHRAPs would have nucleus-localization due to their association with chromatin. We, therefore, wanted to examine if the nucleus-localized proteins were particularly enriched in the top ranked proteins by SILAC ratios. Based on the subcellular localization characterized by Matsuyama et al. [
Top ranked proteins by SILAC ratios are enriched for nucleus-localized proteins and CHRAP-associated functions. (a) Nucleus-localized proteins are enriched in the top 10% ranked proteins by SILAC. Display is identical to Figure
Next, we wanted to know if the CHRAP-associated functions such as chromatin modification and DNA replication and repair would be enriched in the top ranked proteins by SILAC ratios. Based on the gene ontology (i.e., biological process terms), gene functions such as transcription, chromatin modification, DNA replication and repair were over-represented in the top 10% ranked proteins (
In the top 10% ranked proteins by SILAC ratios, we found Psm3, Cbh2, and C27f1.06c that are involved in chromosome organization and chromatin remodeling; Ddb1, Msh3, Spp1, and Uve1 that are involved in DNA replication and repair, and Eri1, C947.08c, Rpb1, and C530.05 that are involved in transcription (see Supplementary Table S2). These proteins would represent a small subset of CHRAPs that are relatively soluble, abundant, and constitutively associated with the chromatin. Deep analysis of CHRAP-prep using SILAC proteomics should allow identifying more proteins with CHRAP-associated functions without prior knowledge.
We also found Prp16, Smb1, and Mug161 that are involved in proteolysis and Ubr1, P8b7.11, and Rpt2 that are involved in mRNA splicing in the top ranked proteins by SILAC ratios. In fact, it is not unusual to find components of the proteolysis machinery that are associated with the chromatin. For instance, the ubiquitin E3 ligase Pcu4 is found to be associated with the RNA-induced transcriptional silencing (RITS) complex involved in heterochromatin assembly [
We noted that some nucleus-localized proteins were not listed in the top 10% ranked proteins by SILAC ratios. Those nucleus-localized proteins might not be associated with the chromatins. To test this possibility, randomly selected 3 top SILAC-ranked proteins Msh3, Prp16, and C18.05c (e.g., their SILAC ratios were 5.86, 4.72, and 4.19 in log 2 scale, resp.) and 2 other proteins Srp2 and Kap95 (e.g., their SILAC ratios are −0.46 and −1.30 in log 2 scale) (see Supplementary Table S2) were subjected to the traditional assay for CHRAPs (see Section
Traditional CHRAP assay to validate SILAC-enriched/depleted proteins. (a) SILAC-enriched nucleus localized proteins are association with the chromatin. Loading quantity of various protein samples such as whole cell extract (W), supernatant-1 (s1) and supernatant-2 (s2; the CHRAP-prep) is indicated in parentheses on the top. Tested proteins and their SILAC ratio in parentheses are indicated at the bottom. (b) SILAC-depleted proteins are unassociated with the chromatin. The display is identical to (a). (c) Subcellular localization of the HA-tagged Srp2 proteins. (d) SILAC-enriched dual-localized proteins are associated with the chromatin. The display is identical to (a).
Some of the dual-localized proteins were found in the top ranked proteins by SILAC ratios (see Supplementary Table S2). To test if they were the true CHRAPs, the 3 randomly selected proteins Uve1, Hsp16, and C530.05 were subjected to the traditional CHRAP assay. The analysis indicated that all 3 proteins exhibited apparent enrichment in the CHRAP-prep when compared to WCE (Figure
Of the 507 proteins identified in the SILAC analysis, 413 were found to be either SILAC enriched (i.e., log 2 SILAC-ratio > 0.585) or depleted (i.e., log 2 SILAC-ratio < −0.585). We wanted to test if the SILAC-enriched proteins have a likelihood of requirement for growth fitness in DNA damage stress, one of the CHRAP-associated functions. For this reason, 188 (~45.5%)
The growth fitness score GFSMMS was proportional to the level of requirement for growth fitness under MMS stress. It was apparent that, among the nucleus-localized proteins, the median GFSMMS of the SILAC-enriched proteins was significantly higher than that of the SILAC-depleted ones (1.43 versus 0.79;
SILAC-enriched nucleus-localized proteins exhibit the high likelihood of requirement for growth fitness under DNA damage stress. The boxplots show the level of growth fitness in various mutant strains. Cells containing a deletion allele of the SILAC-enriched or depleted proteins are indicated. Nucleus-localized (Nuc), dual-localized (Dual), or cytoplasm-localized (Cyto) are shown in (a), (b), or (c), respectively.
We noted that, however, hardly any chromo-domain or bromo-domain containing chromatin remodelers are found in our SILAC analysis of CHRAP-prep. This is probably a result of using the physiological salt concentration in this analysis (see Section
We show that the CHRAP-prep used in traditional assays for CHRAPs is predominated by the abundant cytoplasmic proteins such as ribosomal proteins based on the absolute abundance of proteins. On the other hand, we show that proteomic analysis of CHRAP-prep together with the SILAC-labeled WCE is able to effectively deplete the ribosomal proteins from the top ranked proteins by SILAC ratios. Significantly, we show that the top ranked proteins by SILAC ratios enrich for nucleus-localized proteins that display a high likelihood of requirement for growth fitness under DNA damage stress. Hence, the SILAC-mediated proteomic analysis is capable of determining CHRAPs without prior knowledge. We propose the method shown in this study can be complementary to the proteomic analysis of protein complexes purified via ChIP with TAP-tagged CHRAPs for identification of CHRAP-interacting partners.
Strains used in this study are listed in Table
List of strains used in this studya.
ID | Relevant genotype | Comment |
---|---|---|
LJY3766 |
|
Laboratory stock |
LJY188 |
|
Laboratory stock |
LJY4383 |
|
This study |
LJY4384 |
|
This study |
LJY4385 |
|
This study |
LJY4386 |
|
This study |
LJY3236 |
|
This study |
LJY4224 |
|
This study |
LJY4429 |
|
This study |
LJY4430 |
|
This study |
Note: aBioneer deletion strains used in this study are listed in the Supplementary Table S1.
To enrich the CHRAPs, CHRAP-prep was obtained as described elsewhere with some modification [
A desired amount of proteins was taken and mixed with standard loading buffer for SDS-PAGE analysis. Proteins in gel were electrotransferred onto nitrocellulose membranes for probing with primary antibodies against HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), histone H4 (Upstate Biotechnology, Lake Placid, NY, USA), and
Prior to MS analysis, protein samples were fractionated in SDS-PAGE gels. Gels were sliced into ~50 pieces from top to bottom of a lane. Proteins in gel slices were destained and trypsinized in-gel in 25 mM NH4HCO3 supplemented with 12.5 ng/
Mass spectrometry analysis was performed using a nanoflow high-performance liquid chromatography (HPLC) system (Eksigent, Dublin, CA) connected to a hybrid LTQ-Orbitrap (Thermo Scientific, Bremen, Germany) equipped with a nanoelectrospray ion source (Thermo Scientific). The peptides were separated with a 15 cm long and 75
TPP (Trans-Proteomic Pipeline version 4.2.1, Seattle Proteome Centre, Institute of Systems Biology, Seattle, WA, USA) was used to perform database searching and peptide assignment and validation. For this purpose, all RAW spectra files were converted to mzXML-format. Uninterpreted MS/MS spectra were searched against the
To test whether the CHRAP candidates (e.g., nucleus-localized proteins) could be dominated in the most abundant proteins (i.e., based on the absolute abundance) in the CHRAP-prep, proteomic analysis of the CHRAP-prep proteins without SILAC was performed. In determination of the absolute abundance, the PeptideProphet-qualified peptides (probability ≥ 90%) were quantified by PepQuan and the abundance of proteins was estimated by the median abundance of the respective unique peptides. A total of 376 proteins whose abundance level was approximated by the median level of their unique peptides were listed in Supplementary Table S3. Based on the absolute abundance, nucleus-localized proteins were not overrepresented in the top 10% most abundant proteins (Supplementary Figure S1). Notably, on the other hand, the ribosomal proteins were dominated in the top 10% most abundant proteins, suggesting that the highly abundant non-CHRAP ribosomal proteins are not effectively depleted in the CHRAP-prep.
Of 507 proteins identified in the SILAC-mediated analysis, 413 were either enriched or depleted (i.e., ratio ≥ ±1.5 fold) in CHRAP-prep when compared to WCE. Out of 413 deletion strains, 188 (45.5%) were found in the Bioneer deletion strain collection (Version 1, Bioneer Corporation). Therefore, 188 deletion mutant strains (see Supplementary Table S1) were subjected to minigrowth curve assays using the Bioscreen miniculture growth curve system (Growth Curves USA, Piscataway, NJ) for growth fitness in 1 mM MMS stress. All tests were done in triplicate. Minigrowth curve assay settings used were identical to the previous study [
Growth fitness score in MMS
Binomial test was used to test the nonrandom distribution. In analysis of enrichment or depletion of the nuclear or cytoplasmic proteins, the protein localization information in a genome-wide study by Matsuyama et al. [
Biological process terms were only considered in gene ontology analysis when the occurrence was 15 or greater in the group of 300–500 proteins.
Unpaired two-sample
Original mass spectrometric data are deposited in Tranche database (
Chromatin immunoprecipitation
Chromatin-associated non-histone proteins
Preparation of proteins enriched for CHRAPs
Growth fitness score
Gene ontology
Liquid chromatography
Linear trap quadrupole
Tandem mass spectrometry
Stable isotope labeling with amino acids in cell culture
Whole cell extract.
All authors read and approved the final manuscript. H. Wang, P. Tipthara, K. Tang, and J. Liu participated in research design. H. Wang, and P. Tipthara conducted experiments. SYP contributed new reagents. H. Wang, P. Tipthara, L. Zhu, K. Tang, and J. Liu performed data analysis. J. Liu wrote the paper. H. Wang and P. Tipthara are contributed equally to the paper.
The authors declare that there are no conflict of interests.
The authors are grateful to Dr. J. Li (Nanyang Technological University, Singapore) for his assistance in processing the peptide datasets, Dr. K. Gull (University of Oxford, UK) for the TAT1 antibody, and Dr. A. Lin for the helpful comments. This work was supported by the Agency for Science, Technology, and Research (A-STAR), Singapore to J. Liu and a Nanyang Technological University Ph.D. Studentship to H. Wang.