Enhanced top-down characterization of histone post-translational modifications
© Tian et al.; licensee BioMed Central Ltd. 2012
Received: 28 April 2012
Accepted: 3 October 2012
Published: 3 October 2012
Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.
KeywordsSaltless WCX-HILIC top-down histone posttranslational modification
Histones are important chromatin proteins that act as spools to package and order DNA into structural and manageable chromosomes. Core histones are modified by multiple post-translational modifications (PTMs) such as lysine acetylation, lysine or arginine methylation, and serine or threonine phosphorylation, among others. These PTMs generate a 'histone code'  that is implicated in chromatin-related cellular processes  including transcription , replication , repair , and alternative splicing .
Although core histones comprise only four families (H4, H2B, H2A, and H3), each family has thousands of potential isoforms generated by different combinations of PTMs and protein sequence variation. Traditional antibody-based methods target specific isoforms, typically analyzing one PTM at a time, which makes it virtually impossible to measure combinatorial modifications occurring within the same histone molecule. Recently, high-throughput bottom-up  and middle-down  proteomic methods demonstrated potential for global characterization of PTMs on histone tails. However, these methods are ill-suited for characterizing multiple PTMs dispersed along the entire protein sequence that have been previously discovered to have significant participation in chromatin regulation [2, 9–11].
Top-down proteomic and high-throughput approaches are clearly required to identify and quantify the modulation of multiple intra-molecular histone modifications that synergistically regulate histone functions. Recently, a global top-down study demonstrated the feasibility of intact protein analysis for this purpose by identifying more than 300 histone isoforms using extensive fractionation and customized bioinformatics for global proteome characterization . In histone-focused studies, top-down approaches using an offline two-dimensional liquid chromatography (2D LC) separation and Fourier transform mass spectrometry (FTMS) characterized 34 H4 isoforms from approximately 150 μg of purified H4 protein . However, this study demanded several separations and purification steps for MS compatible samples, requiring a large quantity of starting material and limiting throughput. Clearly, this offline approach is labor-intensive and time-consuming, and requires relatively large sample sizes preventing analysis of biological samples of limited availability such as tumor specimens.
Traditionally, a mobile phase with high-concentration salt in weak cation exchange - hydrophilic interaction LC (WCX-HILIC) has been utilized to separate acetylated  and methylated  histone isoforms. However, the presence of high concentration salts (for example, NaClO4) in the elution buffer leads to ionization suppression and is, therefore, incompatible with modern electrospray ionization (ESI) interfaces typically used for high-throughput online analysis of protein mixtures. Recently, Young et al. successfully developed an alternative 'saltless' pH gradient WCX-HILIC for online middle-down proteomic analysis of human histone H3.2, which enabled an approximately 100-fold reduction in sample requirements and analysis time .Similarly, in this study we utilized a salt-free pH-gradient WCX-HILIC  as the second dimension for separating differentially acetylated/methylated intact protein isoforms within each histone family (H4, H2B, H2A, H3). We combined this separation with online reversed-phase LC (RPLC) in the first dimension to separate histone families and FTMS to enhance MS characterization of intact histones.
In this article, we report a novel high-throughput and high-sensitivity platform for comprehensive characterization of combinatorial histone PTMs at the intact protein level. The novelty stems from use of a metal-free online 2D LC that is coupled with high-performance FTMS. The platform enabled unambiguous identification of 708 histone isoforms from a single analysis of 7.5 μg HeLa core histones.
Results and discussion
Analyses of core histones
Histone isoforms identified from 2D RP-WCX-HILIC LC-MS/MS analysis of 7
Comparison of CID versus ETD
Comparison of CID and ETD for histone isoform identification using 2D RP-WCX-HILIC LC-MS/MS analysis.
Comparison of 2D versus 1D separation
Comparison of this online top-down study with offline top-down, bottom-up, and middle-down studies
While results both presented here and recently published by Tran et al.  are promising in both the field of top-down proteomics and histone analysis, the number of identifications may be influenced by the lack of a histone specific top-down bioinformatics platform. While sequence tags and precursor accurate mass are sufficient for traditional top-down proteomic analyses, histone analysis is complicated by several factors. Such complications include: modification-positional isomers; the small delta mass between acetylation and trimethylation; unknown modifications and those associated with sample processing (that is, oxidation), which could potentially lead to misassignment when searching against databases restricted to known modifications; co-fragmentation of multiple isoforms due to crowding of isotopic distributions in the m/z space; and correct deisotoping. While limiting the search space to previously defined modifications may be required using current tools to complete searches in a realistic time frame (that is, a few days), ultimately, previously unidentified modification sites and forms will not be identified, which brings to light the need for a different type of bioinformatics platform specific to histone analysis. Some of these concerns are addressed by DiMaggio et al. ; however, scaling this middle out tool or others available for the more complicated top-down realm has yet to be achieved. Specific scoring functions are required for ranking the confidence/probability of deisotoped intact mass, site localization of each modification, and protein sequence identification. Additionally, presumably many of the unidentified spectra contain enough fragment ions to assign the correct protein sequence (that is, protein identification), but not sufficient ions to confidently localize PTM site(s) (that is, protein isoform characterization), which is required for comprehensive histone analysis. This gap between protein identification versus characterization will become a larger issue as the popularity of top-down analysis increases, and will hopefully drive the development of a tailored suit of bioinformatics tools for these types of analyses. Co-current optimization of MS technologies/fragmentation methods for histone analysis and bioinformatics platforms that provide confident identifications are needed for comprehensive identifications.
In conclusion, online 2D separation using RP followed by HILIC chromatography allows for the detection and identification of more than seven hundred histone isoforms in a top-down fashion. These results highlight the complexity of histones in general, and demonstrate that modifications that may be important components of the histone code extend well beyond the histone tail region. In general, we envision the metal-free RPLC-WCX/HILIC-FTMS platform being used in a broad range of applications, not only for epigenetic studies of histones, but also for the study of combinatorial PTMs that regulate other classes of proteins.
Materials and methods
The metal-free 2D LC system used in this study is configured as previously reported , except that the system has been further optimized by exchanging the order of the separations and new buffers were developed as described below. A schematic diagram of the new system is shown in Additional file 8. MS grade solvents were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
First dimension RPLC-UV analysis of HeLa core histone mixture
A total of 7.5 μg purified HeLa core histones (Active Motif, Carlsbad, CA, USA) were separated in the first dimension using a Jupiter C5 (5 μm particles, 300 Å pore size) (Phenomenex, Torrance, CA, USA) column (600 mm × 200 μm i.d.) packed in-house. The separation was carried out under constant pressure at 4,000 psi using two Model 100 DM 10,000 psi syringe pumps (with Series D Pump Controller) (ISCO, Lincoln, NE, USA). Mobile phase A consisted of 20% ACN aqueous solution with 5% isopropanol alcohol (IPA) and 0.6% formic acid (FA); mobile phase B consisted of 45% ACN, 45% IPA, and 0.6% FA. The gradient was generated by adding mobile phase B (4,000 psi) to a stirred mixer (volume 2.5 mL equilibrated with 100% mobile phase A at time zero), where an appropriate split flow rate was controlled by the combination of a packed column together with 15 μm i.d. capillary, with an approximate flow of 10 µL/min. Protein elution was monitored online at 214 nm with a SPECTRA100 UV detector (Thermo Separation Products, Waltham, MA, USA). Fractions of interest were collected using two Cheminert column selector systems (VICI, Houston, TX, USA). Once a fraction was collected in one column selector system from the first dimension, the fractionation was switched to the other column selector system and further separation of the first collected fraction in the second dimension ensued.
Second dimension WCX-HILIC-MS/MS analyses of individual histone families
Each histone family fraction was further separated in the second dimension by WCX-HILIC using a PolyCAT A (5 μm particles, 1000 Å pore size) (PloyLC, Columbia, MD, USA) column (50 cm × 100 μm i.d.) packed in house. The separation was carried out with equipment identical to the first dimension mentioned above except for using 70% ACN aqueous solution with 1.0% FA for Mobile phase A and 70% ACN and 8% FA for Mobile phase B. A Cheminert ten-port Nanovolume injection valve (VICI) was used to house two capillary columns, enabling separation and concurrent loading/equilibration between the two columns to increase the throughput of the second dimension. The isolated histone fraction was first loaded onto a solid phase extraction (SPE) column (150 μm i.d. × 5 cm, HILIC stationary phase described above) using Mobile phase A from the second dimension. Once the loading process of one fraction was finished Mobile phase B from the second dimension was added to the mixing vessel to separate the loaded protein and ESI high-resolution MS and MS/MS acquisitions in a LTQ Orbitrap Velos (ThermoFisher Scientific, Waltham, MA) were initiated. ESI voltage was applied by connecting the end of the LC column to a 20 µm i.d. chemically etched capillary emitter with a PEEK union while a voltage was applied through a metal union coupled in the split/purge line out of the analyte path. All acquisitions were performed by the Orbitrap with nominal resolving power of 60,000 (m/z = 400). FTMS MS and MSn automatic gain control (AGC) target values were 1E6 and 3E5, respectively. The number of micro scans for both MS and MSn was three. Fragmentation of precursor ions, isolated with a 1.5 m/z window, was performed by alternating CID (normalized collision energy 35%, 30 ms) and ETD (reaction time 25 ms) for the same precursor ion. Dynamic exclusion was implemented with exclusion duration of 900 s and an exclusion list size of 150. MS/MS was only performed on species with charge states greater than four.
One-dimensional analyses of HeLa core histones using RPLC or WCX-HILIC under the mass spectrometric conditions above were also carried out for the purpose of comparison with two-dimensional analysis.
Change of FDR with different P score cutoff.
P score cutoff
- 2D LC:
two-dimensional liquid chromatography
collision induced dissociation
electron transfer dissociation
false discovery rate
Fourier transform mass spectrometry
solid phase extraction
weak cation exchange - hydrophilic interaction liquid chromatography.
We thank Prof. Neil Kelleher for providing the ProSightPC program. Portions of this work were supported by the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL) Intramural Research and Capability Development Program, the US Department of Energy (DOE) Office of Biological and Environmental Research (OBER), and the NIH National Center for Research Resources (grant RR018522). The research was performed using EMSL, a national scientific user facility sponsored by the DOE-OBER and located at the PNNL. PNNL is a multi-program national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RLO1830.
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