Cryo-electron microscopy structure of the H3-H4 octasome: A nucleosome-like particle without histones H2A and H2B.

The canonical nucleosome, which represents the major packaging unit of eukaryotic chromatin, has an octameric core composed of two histone H2A-H2B and H3-H4 dimers with ∼147 base pairs (bp) of DNA wrapped around it. Non-nucleosomal particles with alternative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Using cryo-electron microscopy, we solved the structure of the H3-H4 octasome, a nucleosome-like particle with a di-tetrameric core consisting exclusively of the H3 and H4 histones. The core is wrapped by ∼120 bp of DNA in 1.5 negative superhelical turns, forming two stacked disks that are connected by a H4-H4' four-helix bundle. Three conformations corresponding to alternative interdisk angles were observed, indicating the flexibility of the H3-H4 octasome structure. In vivo crosslinking experiments detected histone-histone interactions consistent with the H3-H4 octasome model, suggesting that H3-H4 octasomes or related structural features exist in cells.

Tuning the Internal Energy of Ions Produced by Atmospheric and High-Pressure Electrospray by Modulating the Gas Throughput into the First Vacuum Stage.

In a high-pressure environment, electrospray ionization (ESI) can be achieved without discharge between the emitter and the counter electrode, thus enabling the generation of gas-phase ions from liquid with high surface tension, such as pure water, which requires a high onset voltage for stable ESI. In this study, the ion dissociation during the transferring of ions/charged droplets from a superatmospheric pressure environment to vacuum has been systematically investigated using benzyl ammonium thermometer ions. The ion source pressure did not affect the internal energy distribution of ions, whereas the gas throughput into the first vacuum stage clearly influences the internal energy distribution of the ions. The increase in the gas throughput increased the density of molecules/atoms presented in ion transfer/focusing electrodes located in the first vacuum stage. As a result, the mean free path of ions in the first vacuum stage decreases, and the energy of ions decreases by decreasing the kinetic energy involved in each collision between ions and residue gas. The gas throughput into the first vacuum stage is found to describe the internal energy distribution of ions associated with the local conditions more quantitatively instead of using the measured pressure of the vacuum stage, which is different from the effective local pressure. This study also demonstrated the controlled dissociation of ions using the ion transfer settings of the instrument in combination with ion inlet tubes of different sizes.

Development of native mass spectrometry with nanoelectrospray ionization coupled to size exclusion chromatography for proteins.

RATIONALE: Native mass spectrometry (MS) is an analytical technique used to determine the molecular mass of protein complexes without cross-linking. Size exclusion chromatography (SEC) coupled with native MS using conventional electrospray ionization (ESI) has been reported to allow online buffer exchange. To detect a wide variety of protein complexes without a collapse in the ionization process, it is important to build an online system that enables robust analysis with a low flow rate. METHODS: We created an online native MS system equipped with nanoESI connected to the SEC component (online SEC/nanoESI system) and optimized several parameters for SEC separation and ionization. The constructed system was used to measure a solution consisting of a protein mixture of various molecular masses (10-300 kDa) to verify characteristics such as the measurable molecular mass range, reproducibility, and online buffer exchange. RESULTS: The optimal flow rates for SEC separation and nanoESI analysis using this system were 200 and 1 µL/min, respectively. This system was able to analyze proteins in the ranges of 10-300 and 20-300 kDa for protein samples in ammonium acetate and nonvolatile buffer, respectively. Furthermore, the results of consecutive measurements showed that the relative standard deviations of the retention times and observed masses for each protein were sufficiently small. CONCLUSIONS: We created an online SEC/nanoESI system and evaluated its utility for the analysis of various proteins in conventional measurement solvent and nonvolatile buffer. As a result, the structural stability and resolution of the proteins were found to be sufficient when using online buffer exchange. Therefore, this online SEC/nanoESI system would be a useful technique for obtaining mass spectra of various proteins automatically with good resolution, simply by loading samples into an autosampler.

Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and <1 s for the separation. We compared three derivatization methods and found that a method using (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine enables the separation. In addition, we were able to detect dl-2-hydroxyglutaric acid in standard solution at lower concentrations than that previously reported for the serum. These results show the potential of the method to be used in clinical analysis.

Screening of protein-ligand interactions under crude conditions by native mass spectrometry.

A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study. First, overexpressed DHFR with a His-tag was roughly purified with a Ni-sepharose resin and subjected to native mass spectrometry with or without incubation with an inhibitor, Methotrexate (MTX). Even only with the minimum cleanup by the Ni-sepharose resin, intact ions of DHFR-nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR-NADPH-ligand complexes were successfully observed. By optimizing the preparation procedures of the crude sample for native MS, e.g., avoiding sonication for cell lysis, we successfully observed intact ions of the specific DHFR-NADPH-MTX ternary complex starting with cultivation of E. coli in ≤ 25 mL medium. When the crude DHFR sample was mixed with two, four, or eight candidate compounds, only ions of the specific protein-ligand complex were observed. This indicates that the present system can be used as a rapid and convenient method for the rough determination of binding of specific ligands to the target protein without the time-consuming purification of protein samples. Moreover, it is important to rapidly determine specific interactions with target proteins under conditions similar to those in "real" biological systems. Graphical abstract.

Interfacial Dynamics in the Spontaneous Motion of an Aqueous Droplet.

Self-propelled droplets can spontaneously move using chemical energy. In several reports of self-propelled droplets, interfacial chemical reactions occur at the oil/aqueous interface to induce the Marangoni flow. While the dynamics of interfacial tension is essential to the droplet motion, there are few reports that quantitatively discuss the moving mechanism based on interfacial tension measurements. In this study, we focused on the self-propelled motion of an aqueous droplet in the oil phase, where the surfactant monoolein reacts with bromine at the interface, and estimated the physicochemical parameters related to the droplet motion based on the time series of interfacial tension. These results may reveal the general mechanism for the self-propelled motion of aqueous/oil droplets driven by the interfacial chemical reaction.

Structural Diversity of Nucleosomes Characterized by Native Mass Spectrometry.

Histone tails, which protrude from nucleosome core particles (NCPs), play crucial roles in the regulation of DNA transcription, replication, and repair. In this study, structural diversity of nucleosomes was investigated in detail by analyzing the observed charge states of nucleosomes reconstituted with various lengths of DNA using positive-mode electrospray ionization mass spectrometry (ESI-MS) and molecular dynamics (MD) simulation. Here, we show that canonical NCPs, having 147 bp DNA closely wrapped around a histone octamer, can be classified into three groups by charge state, with the least-charged group being more populated than the highly charged and intermediate groups. Ions with low charge showed small collision cross sections (CCSs), suggesting that the histone tails are generally compact in the gas phase, whereas the minor populations with higher charges appeared to have more loosened structure. Overlapping dinucleosomes, which contain 14 histone proteins closely packed with 250 bp DNA, showed similar characteristics. In contrast, mononucleosomes reconstituted with a histone octamer and longer DNA (≥250 bp), which have DNA regions uninvolved in the core-structure formation, showed only low-charge ions. This was also true for dinucleosomes with free DNA regions. These results suggest that free DNA regions affect the nucleosome structures. To investigate the possible structures of NCP observed in ESI-MS, computational structural calculations in solution and in vacuo were performed. They suggested that conformers with large CCS values have slightly loosened structure with extended tail regions, which might relate to the biological function of histone tails.

Stability of the ßB2B3 crystallin heterodimer to increased oxidation by radical probe and ion mobility mass spectrometry.

Ion mobility mass spectrometry was employed to study the structure of the ßB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of ßB3 crystallin across residues 115-126 and 152-166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.

Mass spectrometric approach for characterizing the disordered tail regions of the histone H2A/H2B dimer.

The histone H2A/H2B dimer is a component of nucleosome core particles (NCPs). The structure of the dimer at the atomic level has not yet been revealed. A possible reason for this is that the dimer has three intrinsically disordered tail regions: the N- and C-termini of H2A and the N-terminus of H2B. To investigate the role of the tail regions of the H2A/H2B dimer structure, we characterized behaviors of the H2A/H2B mutant dimers, in which these functionally important disordered regions were depleted, using mass spectrometry (MS). After verifying that the acetylation of Lys residues in the tail regions had little effect on the gas-phase conformations of the wild-type dimer, we prepared two histone H2A/H2B dimer mutants: an H2A/H2B dimer depleted of both N-termini (dN-H2A/dN-H2B) and a dimer with the N- and C-termini of H2A and the N-terminus of H2B depleted (dNC-H2A/dN-H2B). We analyzed these mutants using ion mobility-mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS). With IM-MS, reduced structural diversity was observed for each of the tail-truncated H2A/H2B mutants. In addition, global HDX-MS proved that the dimer mutant dNC-H2A/dN-H2B was susceptible to deuteration, suggesting that its structure in solution was somewhat loosened. A partial relaxation of the mutant's structure was demonstrated also by IM-MS. In this study, we characterized the relationship between the tail lengths and the conformations of the H2A/H2B dimer in solution and gas phases, and demonstrated, using mass spectrometry, that disordered tail regions play an important role in stabilizing the conformation of the core region of the dimer in both phases.

Conclusive evidence of the reconstituted hexasome proven by native mass spectrometry.

It has been suggested that the hexasome, in which one of the H2A/H2B dimers is depleted from the canonical nucleosome core particle (NCP), is an essential intermediate during NCP assembly and disassembly, but little structural evidence of this exists. In this study, reconstituted products in a conventional NCP preparation were analyzed by native electrospray ionization mass spectrometry, and it was found that the hexasome, which migrated in a manner almost identical to that of the octasome NCP in native polyacrylamide gel electrophoresis, was produced simultaneously with the octasome NCP. This result might contribute to understanding the assembly and disassembly mechanism of NCPs.

Gas-phase structure of the histone multimers characterized by ion mobility mass spectrometry and molecular dynamics simulation.

The minimum structural unit of chromatin is the nucleosome core particle (NCP), consisting of 146 bp of DNA wrapped around a histone octamer, which itself contains two H2A/H2B dimers and one (H3/H4)2 tetramer. These multimers possess functionally important tail regions that are intrinsically disordered. In order to elucidate the mechanisms behind NCP assembly and disassembly processes, which are highly related to gene expression, structural characterization of the H2A/H2B dimer and (H3/H4)2 tetramer will be of importance. In the present study, human histone multimers with disordered tail regions were characterized by electrospray ionization (ESI) ion mobility-mass spectrometry (IM-MS) and molecular dynamics (MD) simulation. Experimentally obtained arrival times of these histone multimer ions showed rather wide distributions, implying that multiple conformers exist for each histone multimer in the gas phase. To examine their structures, MD simulations of the histone multimers were performed first in solution and then in vacuo at four temperatures, resulting in a variety of histone multimer structures. Theoretical collision cross-section (CCS) values calculated for the simulated structures revealed that structural models with smaller CCS values had more compact tail regions than those with larger CCS values. This implied that variation of the CCS values of the histone multimers were primarily due to the random behaviors of the tail regions in the gas phase. The combination of IM-MS and MD simulation enabled clear and comprehensive characterization of the gas-phase structures of histone multimers containing disordered tails.

Characterisation of an intrinsically disordered protein complex of Swi5-Sfr1 by ion mobility mass spectrometry and small-angle X-ray scattering.

It is now recognized that intrinsically disordered proteins (IDPs) play important roles as hubs in intracellular networks, and their structural characterisation is of significance. However, due to their highly dynamic features, it is challenging to investigate the structures of IDPs solely by conventional methods. In the present study, we demonstrate a novel method to characterise protein complexes using electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) in combination with small-angle X-ray scattering (SAXS). This method enables structural characterisation even of proteins that have difficulties in crystallisation. With this method, we have characterised the Schizosaccharomyces pombe Swi5-Sfr1 complex, which is expected to have a long disordered region at the N-terminal portion of Sfr1. ESI-IM-MS analysis of the Swi5-Sfr1 complex revealed that its experimental collision cross-section (CCS) had a wide distribution, and the CCS values of the most dominant ions were ∼56% of the theoretically calculated value based on the SAXS low-resolution model, suggesting a significant size reduction in the gas phase. The present study demonstrates that the newly developed method for calculation of the theoretical CCSs of the SAXS low-resolution models of proteins allows accurate evaluation of the experimental CCS values of IDPs provided by ESI-IM-MS by comparing with the low-resolution solution structures. Furthermore, it was revealed that the combination of ESI-IM-MS and SAXS is a promising method for structural characterisation of protein complexes that are unable to crystallise.

Pentafluorobenzylpyridinium: new thermometer ion for characterizing the ions produced by collisional activation during tandem mass spectrometry.

In this study, pentafluorobenzylpyridinium (F5-BnPy+), which has the highest dissociation energy among the reported benzylpyridinium thermometer ion, is proposed to characterize the internal energy distributions of ions activated by higher energy collisional dissociation (HCD) and ion-trap collision-induced dissociation (CID) during tandem mass spectrometry. The dissociation threshold energies of F5-BnPy+ was determined using quantum chemistry calculations at the CCSD(T)/6-311++G(d,p)//M06-2X-D3/6-311++G(d,p) level of theory, and the appearance energies for ion dissociation in HCD and ion-trap CID were estimated using Rice-Ramsperger-Kassel-Marcus theory. The main differences between HCD and ion-trap CID are the collision energies used and the timescales of collisional activation. For both HCD and ion-trap CID, the average internal energy of the ions increased with increasing collision energy. In contrast, the average value for the internal energy of the ions activated by ion-trap CID was lower than that of ions activated by HCD, probably because of the smaller collisional energy and longer activation time of the ion-trap CID experiments. The reported method will aid in the determination of the optimum tandem mass spectrometry parameters for the analysis of small molecules such as metabolites.

Evaluation for Ion Heating of H2A-H2B Dimer in Ion Mobility Spectrometry-Mass Spectrometry.

Ion mobility spectrometry-mass spectrometry (IMS-MS) provides m/z values and collision cross sections (CCSs) of gas-phase ions. In our previous study, an intrinsically disordered protein, the H2A-H2B dimer, was analyzed using IMS-MS, resulting in two conformational populations of CCS. Based on experimental and theoretical approaches, this resulted from a structural diversity of intrinsically disordered regions. We predicted that this phenomenon is related to ion heating in the IMS-MS instrument. In this study, to reveal the effect of ion heating from parameters in the IMS-MS instrument on the conformational population of the H2A-H2B dimer, we investigated the arrival time distributions of the H2A-H2B dimer by changing values of three instrumental parameters, namely, cone voltage located in the first vacuum chamber, trap collision energy (trap CE) for tandem mass spectrometry, and trap bias voltage for the entrance of IMS. These results revealed that the two populations observed for the H2A-H2B dimer were due to the trap bias voltage. Furthermore, to evaluate the internal energies of the analyte ions with respect to each parameter, benzylpyridinium derivatives were used as temperature-sensitive probes. The results showed that the trap CE voltage imparts greater internal energy to the ions than the trap bias voltage. In addition, this slight change in the internal energy caused by the trap bias voltage resulted in the structural diversity of the H2A-H2B dimer. Therefore, the trap bias voltage should be set with attention to the properties of the analytes, even if the effect of the trap bias voltage on the internal energy is negligible.

Differences in the internal energies of ions in electrospray ionization mass spectrometers equipped with capillary-skimmer and capillary-RF lens interfaces.

Small metabolites are commonly analyzed using electrospray ionization mass spectrometry (ESI-MS). Although the protonated form of a compound of interest is typically the target ion in ESI-MS, the protonated forms of small metabolites occasionally undergo fragmentation during ion transmission from ambient conditions to vacuum conditions, hindering the unambiguous identification of analyte molecules. To estimate the fragmentation efficiency during ESI processes, the internal energy distribution of the ions (P(E)) must be evaluated. The common approach for the P(E) evaluation is the survival yield method, which uses thermometer ions. In this study, the P(E) of ions produced by an ESI source in a commercial triple quadrupole mass spectrometer equipped with a capillary-skimmer and capillary-RF lens interfaces was evaluated using benzyl ammonium thermometer ions. Furthermore, this study proposes the use of 3-(aminomethyl)indole and related compounds, which have the lowest Eapp values among the reported thermometer ions, to obtain P(E) values of the ions more accurately. Results showed that P(E) strongly depends on whether a capillary-skimmer interface or capillary-RF lens interface was used for ion transport to the vacuum. ESI-MS with a capillary-skimmer interface provided a considerably lower and narrower P(E) of ions than that with a capillary-RF lens interface, thereby producing intact protonated molecules without significant fragmentation of most small metabolites. However, ESI-MS equipped with capillary-RF lens interfaces provided a higher efficiency of ion transmission than ESI-MS equipped with a capillary-skimmer interface, allowing for highly sensitive analysis of metabolites.

Characterization of the Internal Energy of Ions Produced by Electrospray Ionization Using Substituted Benzyl Ammonium Thermometer Ions.

We propose the use of substituted benzyl ammonium species as thermometer ions to characterize the internal energy distribution of the ions produced by electrospray ionization (ESI). Crucially, we found that the activation of the benzyl ammonium species preferentially provided a benzyl cation via N-Cα bond cleavage. In addition, calculations at the CCSD(T)/cc-PVTZ//M06-2X-D3/6-311++G(d,p) level of theory revealed that the threshold energies of fragmentation of the tested model ions ranged from 86 to 192 kJ mol-1, significantly lower than those of conventional 4-substituted benzylpyridinium thermometer ions. Thus, the substituted benzyl ammoniums are suitable for the characterization of the ESI process under typical experimental conditions. Further, the internal energies of the ions were found to depend on the radiofrequency voltage of the ion funnel, which is used to increase the transport efficiency of the ions from atmospheric to vacuum conditions. Our reported method will aid the determination of the optimum ion-funnel radiofrequency voltage for the analysis of small molecules such as metabolites. Furthermore, benzyl ammoniums are commercially available, which will facilitate the routine and widespread measurement of the internal energy distributions of ions.

Characterization and Value Assignment of a Monoclonal Antibody Reference Material, NMIJ RM 6208a, AIST-MAB.

Monoclonal antibodies have been established as the largest product class of biopharmaceuticals. Since extensive characterization is required for development and quality control of monoclonal antibody, a widely available reference material (RM) is needed. Herein, a humanized IgG1κ monoclonal antibody reference material, RM 6208-a, AIST-MAB, was established by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). The monoclonal antibody solution was produced as a pharmaceutical grade using a Chinese hamster ovary-derived cell line. The assigned indicative value represents the concentration of the antibody with a heterotetrameric structure including oligomeric forms, determined by an amino acid analysis using isotope dilution mass spectrometry, and their homogeneity and stability were assessed. In addition to antibody concentration, various physicochemical properties, including peptide mapping data, charge variants, and aggregates, were examined. This RM is intended for use in validation of analytical procedures and instruments such as a system suitability test for quantification of antibody. It is also intended for comparing and evaluating the results of antibody analyses across analytical methods and analytical laboratories such as inter-laboratory comparison. Both the material and the set of data from our study provide a tool for an accurate and reliable characterization of product quality attributes of monoclonal antibodies in biopharmaceutical and metrology communities.

Fragmentation efficiency of phenethylamines in electrospray ionization source estimated by theoretical chemistry calculation.

Small molecules with polar functional groups, including substituted phenethylamines, are commonly analyzed by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). Analyte molecules are mostly detected in protonated and cation-adducted forms through positive-ion electrospray ionization-mass spectrometry (ESI-MS). However, the ESI of substituted phenethylamines commonly provides an intense signal of fragment ions by ESI in-source collision-induced dissociation (IS-CID), which hinders the unambiguous identification of phenethylamines. This phenomenon was approximated as a unimolecular dissociation model, and the dissociation efficiency was evaluated by various quantum chemistry calculations to determine the ESI IS-CID efficiency. The calculated results were consistent with the experimental data, when the dissociation threshold energy of phenethylamines was calculated using the post-Hartree-Fock (post-HF) method, CCSD(t)/cc-pVTZ//MP2(full)/6-311++G(d,p). In contrast to post-HF methods, the utilization of density functional theory calculations with a suitable functional is recognized as an accurate and competitive low-cost approach. In particular, ωB97-XD, M06-2X-D3, and recently developed Minnesota functionals, such as M11, MN12-SX, and MN15, provided reliable results, as in the case of the post-HF method. The results obtained by the recently developed double hybrid functionals, DSD-PEBP86-D3(BJ), PBE0-DH, and PBE-QIDH, were also reliable. The consideration of ESI IS-CID can facilitate the identification of analyte molecules because most phenethylamines, except for N-methylated analogs, provide an intense signal in the ESI mass spectrum.

Topology and dynamics of melittin within the liposome revealed by a combination of mass spectrometry and chemical modification.

The topology and dynamics of melittin within the liposome were investigated by a mass spectrometry coupled with acetylation. The MALDI-TOF MS and MALDI-QIT-TOF MS/MS analyses revealed that only N-terminal amine of melittin was dominantly acetylated in the presence of liposome although all of four primary amines were completely and rapidly acetylated in aqueous solution. This result indicates that melittin adopts the N-terminal-outside transmembrane topology within the liposome. The time course of acetylation followed the first-order kinetics at any examined temperatures (6-30 degrees C). The rate constant was less than that of the acetylation of melittin in aqueous solution. The activation energy for acetylation (74 kJ mol(-1)) was comparable to that for dissociation of a lipid monomer from the membrane, suggesting a float-like longitudinal motion of melittin within the liposome. These results demonstrate that a mass spectrometry combined with chemical modification is very efficient way for clarifying the topology and dynamics of peptides bound to the membrane.

Mass Spectrometric Characterization of Histone H3 Isolated from in-Vitro Reconstituted and Acetylated Nucleosome Core Particle.

Post-translational modifications (PTMs) of histone N-terminal tails in nucleosome core particle (NCP), such as acetylation, play crucial roles in regulating gene expression. To unveil the regulation mechanism, atomic-level structural analysis of in-vitro modified NCP is effective with verifying the PTMs of histones. So far, identification of PTMs of NCP originating from living cells has mainly been performed using mass spectrometry (MS) techniques, such as bottom-up approach. The bottom-up approach is the most established method for protein characterization, but it does not always provide sufficient information on the acetylated sites of lysine residues in the histone tails if trypsin digestion is carried out. For histone proteins, which have many basic amino acids, trypsin generates too many short fragments that cannot be perfectly analyzed by tandem MS. In this study, we investigated the in vitro acetylation sites in the histone H3 tail using a top-down sequence analysis, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiment, in combination with aminopeptidase digestion. Aminopeptidase can cleave peptide bonds one-by-one from the N-terminus of peptides or proteins, generating N-terminally truncated peptides and/or proteins. As a result, it was identified that this method enables sequence characterization of the entire region of the H3 tail. Also, application of this method to H3 in in-vitro acetylated NCP enabled assigning acetylation sites of H3. Thus, this method was found to be effective for obtaining information on in-vitro acetylation of NCP for structural biology study.