Kazuo Hirayama

Rapid confirmation and revision of the primary structure of bovine serum albumin by ESIMS and frit-FAB LC/MS

Incorrectness of the amino acid sequence of bovine serum albumin (BSA) was suggested from the observed molecular weight of BSA obtained by electrospray ionization mass spectrometry (ESIMS). Lack of a tyrosine residue in the position of 156th was found rapidly, by the combination of frit-fast atom bombardment mass spectrometry/liquid chromatography (Frit-FAB LC/MS), automated Edman degradation and tandem mass spectrometry (MS). Then it turned out that BSA is composed of 583 amino acid residues, and that its average molecular weight is not 66267.1, and it is corrected to 66430.3. Moreover the amino acid sequence of the positions of 94th and 95th was corrected to -QE- by using automated Edman degradation method.

Identification and characterization of oxidized human serum albumin

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases. However, little is known regarding the pathophysiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both the structural and functional differences between reduced and oxidized HSA. Using LC‐ESI‐TOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide‐bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA including protease susceptibility, ligand‐binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions.

Precolumn derivatization reagents for high-speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry.

A rapid analytical method for amines and amino acids was developed, involving derivatization with the novel reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), followed by reversed-phase high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). More than 100 different analytes with amino groups, including amino acids in biological fluids such as mammalian plasma, could be measured within 10 min. The analytes were easily derivatized with APDS under the mild conditions. Selective reaction monitoring of ESI-MS/MS in positive mode was carried out to include the transitions of all of the protonated molecular ions of analytes derivatized with APDS to the common fragment at m/z 121, which was derived from the amino pyridyl moiety of the reagent. We evaluated the retention time precision, the quantification limits, the linearity, the intra- and inter-day precisions and the accuracy of 22 typical amino acids found in biological fluids, by analyzing a standard amino acid mixture and rat plasma. The intra-day relative standard deviations (RSDs) of the retention times of the 22 amino acids and their internal standards were within 0.9% and the inter-day RSDs were less than 1.1%, except for asparagines, with an RSD of 1.9%. The intra-day and inter-day RSDs of amino acid analyses in rat plasma were within 8.0% and 4.5%, respectively. The method, which facilitates the amino acid analysis of more than 100 samples in a day, represents an alternative to traditional amino acid analysis techniques, such as chromatography using postcolumn derivatization by ninhydrin.

COMPARATIVE STUDY OF TOXIC AND NON-TOXIC CYANOBACTERIAL PRODUCTS : NOVEL PEPTIDES FROM TOXIC NODULARIA SPUMIGENA AV1

Abstract Two types of novel peptides, cyclic peptides, nodulapeptins A (1) and B (2) and linear peptides, spumigins A∼C (3∼6), were isolated together with nodularin from toxic Nodularia spumigena AV1. Their structures were determined by 2D-NMR techniques, the advanced Marfeys method and MS/MS experiments.

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry

An automated method for high-throughput amino acid analysis, using precolumn derivatization high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home-built auto-sampler system. Amino acids were derivatized with 3-aminopyridyl-N-hydroxysuccinimidyl carbamate, and a 3 microm Wakosil-II 3C8-100HG column (100 x 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra- and inter-precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%.

Mutual separation and preconcentration of vanadium(V) and vanadium(IV) in natural waters with chelating functional group immobilized silica gels followed by determination of vanadium by inductively coupled plasma atomic emission spectrometry

The mutual separation and preconcentration of VV and VIV using chelating functional group immobilized silica gels (CISs) has been studied, based on inductively coupled plasma atomic emission spectrometric detection. Ethylenediamine-bonded (ED–CIS) and ethylenediaminetriacetate-bonded (ED3A-CIS) silica gels were used. A two-column system consisting of a first column of ED–CIS and a second column of ED3A-CIS was developed for on-line separation and preconcentration of VV and VIV. In the pH range 2.5–3.0, ED–CIS retains VV and separates it completely from VIV, whereas ED3A-CIS collects both VV and VIV. The second column can be used to preconcentrate VIV in the breakthrough solution from the first column. Separation is achieved with recoveries of 91–105%. The recovery of both ions is 98% or better. An enrichment factor of 40 is obtained, with which the detection limit of V with inductively coupled plasma atomic emission spectrometry is improved down to 60 pg ml–1. The method was applied successfully to V speciation in natural waters.

Comparative study of toxic and non-toxic cyanobacterial products: A novel glycoside, suomilide, from non-toxic Nodularia spumigena HKVV

Abstract A novel glycosidic compound, suomilide ( 1 ), was isolated together with 1-( O - α -glucopyranosyl)-3,25-hexacosanediol, a “heterocyst glycolipid” from the non-toxic Nodularia spumigena HKVV. Their structures were determinated by 2D-NMR techniques and MS/MS experiments.

Saxitoxin as a toxic principle of a freshwater puffer, Tetraodon fangi, in Thailand

Saxitoxin was identified in a freshwater puffer, Tetraodon fangi, which caused food poisoning in Thailand. Tetrodotoxin, a puffer toxin, was not detected in the species by the HPLC-fluorometric analysis, showing that tetrodotoxin is absent or under any detectable level. The result of this study shows that saxitoxin can be a major toxin in puffer.

Structural studies of the Maillard reaction products of a protein using ion trap mass spectrometry.

The early stage products of the Maillard reaction of egg white lysozyme with D-glucose were studied. Incubation with D-glucose at 50 degrees C for 20 days caused reaction on the Lys and Arg residues of lysozyme as follows: all of the six Lys residues and 10 of the 11 Arg residues in lysozyme reacted with D-glucose; Arg 61 did not react with D-glucose. The Lys residues reacted with D-glucose with 1 mol of dehydration per mole of residue, and the Arg residues reacted with 2 mol of dehydration per mole of residue. The major constituent of the Amadori product with the epsilon-amino group of the Lys residue and the D-glucose was found to be the beta-pyranose form. The structure of the early stage product of the Maillard reaction of a protein with a sugar is the same as that of an amino acid with a sugar.

A single CTL clone can recognize a naturally processed HIV‐1 epitope presented by two different HLA class I molecules

Although it is known that a single peptide can be recognized by CTL restricted to two MHC class I alleles, there is no direct evidence for presentation of a single peptide by two MHC class I molecules. Furthermore, it is unclear whether such peptides are presented to the same T cell or to different T cells. Our previous study suggested that CTL recognition of the human immunodeficiency virus‐1 (HIV‐1) Pol HIV‐B35‐SF2‐24 epitope (IPLTEEAEL) occurs via both HLA‐B35 and HLA‐B51 restriction. Here we provide the first direct evidence that a single CTL clone can recognize this peptide presented by both HLA‐B35 and HLA‐B51. Furthermore, we directly purified this peptide eluted from both HLA‐B*3501 and HLA‐B*5101 molecules isolated from target cells infected with HIV‐1 recombinant vaccinia virus. These results demonstrate that HIV‐B35‐SF2‐24 is a naturally processed peptide which is presented by both HLA‐B*3501 and HLA‐B*5101. TCR analysis of one CTL clone suggested that it is a single clone. B*3501‐SF2‐24‐tetrameric complexes inhibited both HLA‐B*3501‐ and HLA‐B*5101‐restricted recognition of this clone, suggesting that the TCR of this clone cross‐recognize the structure of both HLA class I‐peptide complexes.