Megumi Kato

Application of amino acid analysis using hydrophilic interaction liquid chromatography coupled with isotope dilution mass spectrometry for peptide and protein quantification

Amino acid analysis that is based on the use of hydrophilic interaction liquid chromatography (HILIC) coupled with isotope dilution mass spectrometry (IDMS) has been developed for the accurate quantification of underivatized amino acids from hydrolyzed protein/peptide. Sufficient separation of amino acids on a zwitterion chromatography (ZIC)-HILIC column was achieved after removal of chloride ions in the hydrolyzate. The detection limits and quantification limits as concentration of the four amino acids ranged from 0.003 to 0.04pmol microL(-1) and from 0.01 to 0.1pmol microL(-1), respectively. The analytical results for the certified reference materials, angiotensin I and bovine serum albumin (BSA), were satisfactory. Furthermore, the quantitative results by this method were compared with those by the commercially available precolumn method, derivatizd with aminoquinolylhydroxysuccinimidyl carbamate (AQC method), and better recovery and more precise data were obtained with this method.

Sequence Structure of Human Nucleosome DNA

Abstract Positional distributions of various dinucleotides in experimentally derived human nucleosome DNA sequences are analyzed. Nucleosome positioning in this species is found to depend largely on GG and CC dinucleotides periodically distributed along the nucleosome DNA sequence, with the period of 10.4 bases. The GG and CC dinucleotides oscillate counterphase, i.e., their respective preferred positions are shifted about a half-period from one another, as it was observed earlier for AA and TT dinucleotides. Other purine-purine and pyrimidine-pyrimidine dinucleotides (RR and YY) display the same periodical and counterphase pattern. The dominance of oscillating GG and CC dinucleotides in human nucleosomes and the contribution of AG(CT), GA(TC), and AA(TT) suggest a general nucleosome DNA sequence pattern—counterphase oscillation of RR and YY dinucleotides. AA and TT dinucleotides, commonly accepted as major players, are only weak contributors in the case of human nucleosomes.

Dinucleosome DNA of Human K562 Cells: Experimental and Computational Characterizations

Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.

Evolutionary conservation and functional synergism of curved DNA at the mouse epsilon- and other globin-gene promoters.

Human and mouse globin genes were separated approximately 200 million years ago but still share homology and synergism in many aspects including DNA structure. We first mapped DNA bend sites in the mouse ε-globin gene and found that these sites were distributed in a regular manner except in the coding region and their overall average distance was 650.7 bp. The first bend site upstream of the cap site (MεB-1, −334 to −147 bp) was found to contain A+T-rich sequences and features contributing to DNA curvature by computer analysis. Transcription assays using deletion constructs indicated strong promoter activity up to bp −215 in erythriod K562 cells. Therefore, the MεB-1 site was located immediately upstream of the promoter region. A reporter gene assay using a series of constructs containing the promoter region revealed that the MεB-1 site showed repressor activity, and on replacement of the DNA curvature with one from another source the activity was retained. A similar feature was found in the other conserved B-1 sites in the human, bovine, and rabbit β-like globin genes, with the exception of an unconserved B-1 site in the chicken β-globin gene. A common feature of these conserved B-1 sites was not the nucleotide sequences but the DNA curvature. Furthermore, a unique nucleosome phase at the MεB-1 site was likely to be directed by DNA curvature. Based on these results, DNA curvature is one of the major features of these promoter regions which might influence transcription through nucleosome positioning.

Development of High-purity Certified Reference Materials for 17 Proteinogenic Amino Acids by Traceable Titration Methods

To ensure the reliability of amino acid analyses, the National Metrology Institute of Japan of the National Institute of Advanced Industrial Science and Technology (NMIJ/AIST) has developed high-purity certified reference materials (CRMs) for 17 proteinogenic amino acids. These CRMs are intended for use as primary reference materials to enable the traceable quantification of amino acids. The purity of the present CRMs was determined based on two traceable methods: nonaqueous acidimetric titration and nitrogen determination by the Kjeldahl method. Since neither method could distinguish compounds with similar structures, such as amino acid-related impurities, impurities were thoroughly quantified by combining several HPLC methods, and subtracted from the obtained purity of each method. The property value of each amino acid was calculated as a weighted mean of the corrected purities by the two methods. The uncertainty of the property value was obtained by combining measurement uncertainties of the two methods, a difference between the two methods, the uncertainty from the contribution of impurities, and the uncertainty derived from inhomogeneity. The uncertainty derived from instability was considered to be negligible based on stability monitoring of some CRMs. The certified value of each amino acid, property value with uncertainty, was given for both with or without enantiomeric separation.

Amino acid analysis by hydrophilic interaction chromatography coupled with isotope dilution mass spectrometry.

Here, we describe an amino acid analysis that is based on the use of hydrophilic interaction liquid chromatography coupled with isotope dilution mass spectrometry for the accurate quantification of underivatized amino acids from hydrolyzed peptide/protein. Twelve underivatized amino acids were separated and detected during an 88-min runtime. The absolute limits of detection and limits of quantification (on column) of the four amino acids (isoleucine, phenylalanine, proline, and valine) were in the range of 6-80 and 20-200 fmol, respectively. As little as 25 pmol of peptide or protein hydrolysates is sufficient for determining absolute content.

Effects of the pH and Concentration on the Stability of Standard Solutions of Proteinogenic Amino Acid Mixtures

To prepare metrologically traceable amino acid mixed standard solutions, it is necessary to determine the stability of each amino acid present in the mixed solutions. In the present study, we prepared amino acid mixed solutions using certified reference standards of 17 proteinogenic amino acids, and examined the stability of each of these amino acids in 0.1 N HCl. We found that the concentration of glutamic acid decreased significantly during storage. LC/MS analysis indicated that the instability of glutamic acid was due to the partial degradation of glutamic acid to pyroglutamic acid in 0.1 N HCl. Using accelerated degradation tests, we investigated several solvent compositions to improve the stability of glutamic acid in amino acid mixed solution, and determined that the change of the pH by diluting the mixed solution improved the stability of glutamic acid.

Determination of Sulfur in Bio-Samples by ICP-QMS/QMS with an ORC

A method for determination of sulfur was investigated using an inductively coupled plasma tandem quadrupole mass spectrometer (ICP-QMS/QMS) with an octopole reaction/collision cell (ORC). It helped to achieve a lower background equivalent concentration (BEC) and a lower detection limit (DL) when 10% HNO3 with 10% ethanol was applied as the rinse solution. Sulfur measurement was carried out by shifting the measured mass from 32S+ to 32S16O+ by reaction with O2 gas. The introduction of H2 together with O2 as reaction gases provided a lower DL. The BEC and DL of 32S were 0.83 ng g-1 and 0.03 ng g-1, respectively. The validation of the present method was confirmed by determining sulfur in certified reference materials, NIST SRMs 2773, 2298, and 2299. The results of sulfur in amino-acids and protein indicate that determination of sulfur could be an effective technique for quantification of sulfur-containing amino acids and proteins.