Noriaki Ishioka

Optical resolution of phenylthiohydantoin-amino acids by capillary electrophoresis for protein sequencing

Abstract An advanced method is described for the complete separation of phenylthiohydantoin (PTH)- dl -amino acids for protein sequencing. Optical resolution of all standard PTH- dl -amino acids was successfully developed using some chiral selectors, although the resolution of only PTH- dl -His and Lys could not be confirmed, due to low reproducibility and the presence of impurities. In addition, mixed chiral selectors for making a single electrolyte with the ability to optically resolve all standard PTH- dl -amino acids were investigated. Using the only resulting electrolyte, sequence determination of [ d -Ala2]-methionine enkephalin, with dl differentiation, was performed.

Optical resolution of phenylthiohydantoin-amino acids and identification of phenylthiohydantoin-d-amino acid residue of [d-Ala2]-methionine enkephalin by capillary electrophoresis

Abstract We propose a system of protein sequence analysis with dl differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE. After fractionation of phenylthiohydantoin (PTH)-amino acids from the protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, o -trimethyl-β-cyclodextrin (TM-β-CD) and others. In addition, optical resolution of all standard PTH- dl -amino acids including PTH- dl -carboxymethyl-Cys (CM-Cys) and cysteic acid (CYA) except for PTH- dl -Lys was successfully developed. The resolution of PTH- dl -Lys could not be reconfirmed due to low reproducibility and the impurities. As a model peptide, [ d -Ala 2 ]-methionine enkephalin ( l -Tyr– d -Ala–Gly– l -Phe– l -Met), was used and the sequence with dl differentiation was completely determined.

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.

Identification of Chirality of Phenylthiohydantoin-D-Amino Acid Residue of [D-ala2]-Metthionine Enkephalin by Capillary Electrophoresis: Suppression and Control of Racemization Ratio in the Edman Sequencing Method

Abstract This paper describes the suppression and control of the racemization ratio (D or L/D+L) of phenylthiohydantoin (PTH) amino acids in the Edman sequencing method. Most of the racemization occurs in the cyclization/cleavage step. Although optimization of partial racemization using a mixture of TFA and boron trifluoride (BF3)-ethyl ether complex, which is effective in suppressing racemization in the cyclization/cleavage reaction. The partial racemization in PTH derivatization is often useful for DL differentiation, because a minor L- or D-peak produced by racemization can be used as an internal standard in CE. Using the partial racemization method with mixed acids as a cyclization/cleavage reagent, the sequence determination of [D-Ala2]-methionine enkephalin, with DL differentiation, was achieved on a sequencer.

Detection of a genetic variant, lysine→glutamic acid at position 372 of human serum albumin, by capillary electrophoresis and structural identification

A genetic variant of human serum albumin (alloalbumin) is detected by capillary electrophoresis (CE). Two albumin peaks, which were in the ratio of approximately one, were clearly separated. One of the peaks had the same migration time as normal albumin (Alb A) and the other (Alb X) had a longer migration time. SDS-polyacrylamide gel electrophoresis of CNBr fragments (CB) of Alb X indicated that the amino acid substitution was localized in the CB5 fragment (residue 330-446) of the molecule, because of anomalous migration of CB5 in the gel. The CE mapping of the tryptic peptides from the variant CB5 revealed clearly the existence of a new peptide, and the lack of two normal peptides. The sequence analysis of the variant peptide collected by CE micropreparation showed that the N-terminus of the variant peptide corresponded to that of T49 in Alb A. The substitution site, lysine-->glutamic acid at the position 372, was revealed by sequence determination of the variant peptide purified by reversed-phase HPLC.

Analysis of Con A-binding glycoproteins in synaptosomal membranes

Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pI5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family.