Nec Corporation

Tokyo Rikakikai Co., Ltd.

Akira Tsugita

Kenichi Kamijo

Kenji Miyazaki

Takuji Nabetani

Hiroaki Torii

Hirotaka Minagawa

Naoyuki Takahashi

Toshimasa Yamazaki

Ichiro Ishida

The present invention provides a method for analyzing the C-terminal amino acid sequence of a peptide by using a reaction for successively releasing the C-terminal amino acids of the peptide, which method can suppress, when successively releasing the C-terminal amino acids of a peptide of long amino acid length, such a undesirable side reaction as cleavage of peptide bond in the intermediate position of the peptide and can carry out the chemical treatment thereof under widely applicable conditions; In the method, a dry sample of a peptide with long amino acid length is beforehand subjected to an N-acylation treatment; by using a reaction reagent where an alkanoic acid anhydride is combined with a small amount of a perfluoroalkanoic acid, successive release of C-terminal amino acids is conducted under mild conditions; a hydrolysis treatment is applied; then, selective fragmentization at site of arginine residue is performed by digestion by trypsin; thereafter, decreases in molecular weight are measured for the C-terminal side fragments derived from a series of reaction products with use of a MALDI-TOF-MS apparatus; thereby, the C-terminal amino acid sequence of the peptide sample is identified.

Method for analyzing C-terminal amino acid sequence of peptide using mass spectrometry

The present invention provides, as a method of analyzing the C-terminal amino acid sequence of a peptide with use of reaction technique for successively releasing the C-terminal amino acids, in which undesirable side reactions, such as cleavage of a peptide bond at the middle of the peptide, can be prevented and chemical treatments therein can be carried out under widely applicable conditions in the course of successive release of the C-terminal amino acids from a peptide, such a method comprising steps of dehydrating the gel on which a target peptide that has been separated by gel electrophoresis is held in the bound state; immersing it in a mixture solution of an alkanoic acid anhydride added with a small amount of a perfluoroalkanoic acid in a dipolar aprotic solvent to re-swell the gel carrier, forming a 5-oxazolone structure, at a temperature chosen in the range of from 30&#xb0; C. to 80&#xb0; C., followed by the cleavage of the 5-oxazolone ring to release the C-terminal amino acids, and then specifying the C-terminal amino acid sequence of the peptide based on the measured decrease in the molecular weight of a series of reaction products resulting therefrom.

Method of analyzing C-terminal amino acid sequence of peptide

The present invention provides, as for a method for analyzing the C-terminal amino acid sequence of a peptide by using a reaction for successively releasing the C-terminal amino acids of the peptide, which method can suppress, when successively releasing the C-terminal amino acids of a peptide of long amino acid length, such a undesirable side reaction as cleavage of peptide bond in the intermediate position of the peptide and can carry out the chemical treatment thereof under widely applicable conditions, a following method wherein a dry sample of a peptide with long amino acid length is beforehand subjected to an N-acylation treatment; by using a reaction reagent where an alkanoic acid anhydride is combined with a small amount of a perfluoroalkanoic acid, successive release of C-terminal amino acids is conducted under mild conditions; a hydrolysis treatment is applied; then, selective fragmentization at site of arginine residue is performed by digestion by trypsin; thereafter, decreases in molecular weight are measured for the C-terminal side fragments derived from a series of reaction products by analysis in negative mode of a MALDI-TOF-MS apparatus; thereby, the C-terminal amino acid sequence of the peptide sample is identified.

Method of analyzing c-terminal amino acid sequence of peptide

An analyte peptide is selectively degraded sequentially by using an alkanoic anhydride (S). The original peptide and a series of degradation reaction products having peptide in which one or more C-terminal-sided amino acids are deleted, are subjected to a certain posttreatment (S). The molecular weight of the reaction products is measured by mass spectrometry (S). And, the amino acid sequence of the original peptide from C-terminal is determined, based on the molecular weight obtained by mass spectrometry (S).

The present invention provides a method for analyzing the C-terminal amino acid sequence of a peptide by applying reaction technique for successively releasing the C-terminal amino acids therefrom, which method can suppress, when releasing the C-terminal amino acids of the peptide in sequence, such as an undesirable side reaction as cleavage of peptide bond in the intermediate position of the peptide, and allows to carry out the chemical treatment thereof under widely applicable conditions. In the method according to the present invention, an alkanoic acid anhydride and a perfluoroalkanoic acid both of vapor phase, which are supplied from a mixture containing an alkanoic acid anhydride with a small amount of a perfluoroalkanoic acid added thereto, are allowed to act on a dry sample of the peptide to be examined in a dry atmosphere at a temperature chosen in a range of 15 to 60&#xb0; C.; whereby the release of the C-terminal amino acid is resulted from successive formation of a 5-oxazolone structure being followed by cleavage of the 5-oxazolone ring; and then the C-terminal amino acids sequence is identified by analysis based on the decrease in molecular weight in a series of the reaction products obtained.

Method of analyzing peptide for determining C-terminal amino acid sequence

In a method for efficiently analyzing a posttranslational modification of a protein using no enzyme, a protein or peptide to be analyzed is reacted with an acid (a thioester or hydrazine) under certain conditions. This makes it possible to detect variously modified states of a protein or peptide, whereby the identification of each specific modifying group and the position of each modified amino acid can be efficiently analyzed using a chemical method and a mass spectrometric apparatus.

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Tokyo, Japan

Akira Tsugita