Patent ID: 7879616
Filing Date: 2011-02-01
Classification: G01N

Abstract:
1. A method for analyzing, by means of mass spectrometry, the C-terminal amino acid sequence of a peptide to be examined, which method comprises the following steps: a step of preparing a mixture containing a series of reaction products that are obtained from an original peptide to be examined by releasing the C-terminal amino acids successively by chemical means and the remains of the original peptide, a step of analyzing the differences in molecular weight between said series of reaction products and the original peptide by means of mass spectrometry to measure the decreases in molecular weight associated with the successive release of the C-terminal amino acids, and a step of identifying a series of the amino acids removed successively, based on a series of the measured decreases in molecular weight and arranging them from the C-terminus to obtain the information of the C-terminal amino acid sequence of the peptide, wherein said process for releasing the C-terminal amino acids successively comprises at least the following steps: a pretreatment step for providing the protection by means of N-acylation in which N-acylation with the acyl group derived from the alkanoic acid anhydride is applied to the N-terminal amino group of the peptide as well as to the amino group on the side chain of the lysine residue which may be included in the peptide, a step of allowing an alkanoic acid anhydride to act on the dry sample of said peptide to be examined after N-acylation protection in the presence of a catalytic amount of a perfluoroalkanoic acid to release the C-terminal amino acids in association with a process that at the C-terminus of the peptide, the formation of a 5-oxazolone structure represented by the following general formula (III): wherein R1 is a side chain of the C-terminal amino acid of the peptide and R2 is a side chain of the amino acid residue positioned just before the C-terminal amino acid, is followed by the cleavage of the 5-oxazolone ring, wherein the formation of the 5-oxazolone structure and the cleavage of the 5-oxazolone ring is carried out in parallel at the same temperature by using the alkanoic acid anhydride in the presence of the catalytic amount of the perfluoroalkanoic acid to successively release the C-terminal amino acids, and a hydrolysis treatment step which comprises applying, to a mixture containing a series of reaction products obtained in said step of releasing the C-terminal amino acids successively, a post-treatment of removing said remaining alkanoic acid anhydride and perfluoroalkanoic acid therefrom, and then allowing water molecules to act thereto in the presence of a catalytic amount of a basic, nitrogen-containing, aromatic compound or a tertiary amine compound to give rise to a hydrolysis reaction, wherein the hydrolysis reaction is carried out by using the water molecules in the presence of a catalytic amount of the basic, nitrogen-containing, aromatic compound or the tertiary amine compound, whereby the mixture containing the series of reaction products having a carboxyl group at their C-terminal as well as the remains of the original peptide having a carboxyl group at its C-terminus are prepared as a sample to be used for the analysis by means of MALDI-TOF-MS, wherein said step of measuring the decreases in molecular weight associated with the successive release of the C-terminal amino acids employs a technique which comprises: allowing trypsin to act on the sample in a buffer solution to carry out the treatment for the enzymatic digestion specific to trypsin of said peptide chain which holds N-acylation protection as for the N-terminal amino group of the peptide chain as well as to the amino group on the side chain of the lysine residue that may be contained in the peptide chain, and thereby, conducting selective cleavage of the C-terminal side peptide bond of each arginine residue that present in the peptide chain to complete peptide fragmentization, applying a desalting treatment to remove the buffer solution component, followed by recovering and drying the peptide fragments after the digestion treatment by trypsin, next, conducting, as for the dried mixture containing said peptide fragments recovered after the digestion treatment by trypsin, molecular weight measurement for the cationic species of (M+H) with respect to the corresponding mass spectra of the ion species, which are measured in said molecular weight measurement for the cationic species of (M+H) judging that the peaks of the peptide fragments each having an arginine residue at the C-terminus, which fragments are produced by said digestion treatment by trypsin, are peaks that give such intensities that the intensity in the molecular weight measurement for the cationic species of (M+H) based on a series of the peaks that gives a relatively larger intensity in the molecular weight measurement for the anionic species of (M−H) wherein the step of analysis of the spectra, in which the range to be analyzed for the analysis operation of spectra is selected within m/z value of 4,000 or less, comprises the following Steps 1 to 9: (Step 1) a step for identification of internal standard peaks derived from trypsin, which comprises: with respect to the peptide fragments derived from the autolysis of trypsin having a known molecular weight, which are concomitant with the digestion treatment with trypsin used for peptide fragmentization, and incorporated into the dry mixture containing the peptide fragments, identifying the peaks of the cationic species of (M+H) then, identifying the peaks of the corresponding anionic species of (M−H) (Step 2) a step for identification of major ion species peaks, which comprises: excluding said peaks assigned for the cationic species leaks derived from trypsin from the result of molecular weight measurements for cationic species, identifying the highest peak of cationic species having the highest peak intensity, in a m/z value range of 4,000 to 500, and by using the peak intensity of the highest peak of cationic species as a basis, selecting peaks of cationic species each having a peak intensity of 1/40 or more relative to the basis to make up the first group of cationic species peaks therewith, next to that, excluding said peaks assigned for the anionic species peaks derived from trypsin from the result of molecular weight measurements for anionic species identifying the highest peak of anionic species having the highest peak intensity, in a m/z value range of 4,000 to 500, and by using the peak intensity of the highest peak of anionic species as a basis, selecting anionic species peaks each having a peak intensity of 1/40 or more relative to the basis to make up the first group of anionic species peaks therewith, (Step 3) a step for identification of counter ion species peaks for the major ion species peaks, which comprises: identifying, in the result of the molecular weight measurements for anionic species, peaks due to anionic species each corresponding to each peak of said first group of cationic species peaks to make up the second group of anionic species peaks therewith, next to that, identifying, in the result of the molecular weight measurements for cationic species, peaks due to cationic species each corresponding to each peak of said first group of anionic species peaks to make up the second group of cationic species peaks therewith, (Step 4) a step for identification of major ion species peaks having significant counter-ionic species, which comprises: making up the overlapping group between the first group of anionic species peaks and the second of anionic species peaks to define it as the third group of anionic species peaks, and also making up the sum group of the first group of anionic species peaks and the second group of anionic species peaks to define it as the fourth group of anionic species peaks, next to that, making up the overlapping group between the first group of cationic species peaks and the second group of cationic species peaks to define it as the third group of cationic species peaks, and also making up the sum group of the first group of cationic species peaks and the second group of cationic species peaks to define it as the fourth group of cationic species peaks, with respect to each cationic species peak corresponding to each peak of said third group of anionic species peaks, calculating the relative peak intensity on the basis of said peak intensity of the highest cationic species peak identified at the Step 2 and, with respect to each anionic species peak of said third group of anionic species peaks, calculating the relative peak intensity on basis of said peak intensity of the highest anionic species peak identified at the Step 2, and comparing the two relative peak intensities with each other, identifying those corresponding cationic species peaks each having a relative intensity which is 3/2 or more relative to that of the peak of the third group of anionic species peaks to make up the fifth group of cationic species peaks therewith, meanwhile, identifying those anionic species peaks each having a relative intensity which is 3/2 or more relative to that of the corresponding cationic species peaks to make up the fifth group of anionic species peaks therewith, (Step 5) a step for identification of major ion species peaks caused by peptide fragments derived from target peptide to be analyzed, which comprises: based on the m/z value of each cationic species peak of the fourth group of cationic species peaks, calculating the differences in the m/z value between each adjacent peaks thereof, meanwhile based on the m/z value of each anionic species peak of the fourth group of anionic species peaks, calculating the differences in the m/z value between each adjacent peaks thereof, with respect to each peak of the fifth group of cationic species peaks, examining said peak as to the following criteria: (5a-1) a cationic species peak having a m/z value smaller than the m/z value of said peak by the molecular weight of 18 corresponding to loss of one water molecule is present in the fifth group of cationic species peaks; (5a-2) a cationic species peak having a m/z value larger than the m/z value of said peak by the molecular weight excess equivalent to the formula weight of the acyl group used for said N-acylation protection is present in the fifth group of cationic species peaks and (5a-3) a cationic species peak having a ink value larger than the m/z value of said peak by the combination of the molecular weight decrease of 18 corresponding to loss of one water molecule and excess equivalent to the formula weight of the acyl group used for said N-acylation protection is present in the fifth group of cationic species peaks; to select those cationic species peaks each satisfying at least one of said requirements (5a-1) to (5a-3), and then making up the sixth group of cationic species peaks therewith, meanwhile, with respect to each peak of the fifth group of anionic species peaks, examining said peak as to the following criteria: (5b-1) an anionic species peak having a m/z value smaller than the ink value of said peak by the molecular weight of 18 corresponding to loss of one water molecule is present in the fifth group of anionic species peaks; (5b-2) an anionic species peak having a m/z value larger than the m/z value of said peak by the molecular weight excess equivalent to the formula weight of the acyl group used for said N-acylation protection is present in the fifth group of anionic species peaks; and (5b-3) an anionic species peak having a ink value larger than the m/z value of said peak by the combination of the molecular weight decrease of 18 corresponding to loss of one water molecule and excess equivalent to the formula weight of the acyl group used for said N-acylation protection is present in the fifth group of anionic species peaks; to select those anionic species peaks each satisfying at least one of said requirements (5b-1) to (5b-3), and then making up the sixth group of anionic species peaks therewith, judging that the seventh group of cationic species peaks are a group of cationic species peaks caused by peptide fragments per se derived from the target peptide to be analyzed, and judging also that the seventh group of anionic species peaks are a group of anionic species groups caused by peptide fragments per se derived from the target peptide to be analyzed, (Step 7) a step for identification of peptide fragments each having arginine at the C-terminus of its peptide chain, produced by the digestion treatment by trypsin, which comprises: selecting each anionic species peak which corresponds to each cationic species peak of the seventh group of cationic species peaks, from the peaks being present in the fourth group of anionic species peaks, to make up the eighth group of anionic species peaks therewith, with respect to each peak of the eighth group of anionic species peaks, selecting group of anionic species peaks of which a m/z value difference from the fiducial m/z value of the said anionic species peak is found within the range of less than 200, based on set of the m/z value differences between adjacent peaks that are calculated in the Step 5, from the peaks being present in the fourth group of anionic species peaks, and confirming that there is not present, in the thus-selected groups, any peak whose m/z value difference between those peaks is equivalent to the formula weight of natural chain a-amino acid residue: —NH—CH(R)—CO— (R is a side chain of said amino acid residue) or of a-amino acid residue protected by acylation wherein the hydroxy group or amino group of its side chain is modified by substitution with the acyl group used in said N-acylation protection, and thus judging that said eighth group of anionic species peaks are the group of anionic species peaks from peptide fragments each having arginine at the C-terminus of its peptide chain, which are derived from the target peptide to be examined and produced by the treatment for digestion by trypsin, judging that the sixth group of cationic species peaks selected thereby are a group of cationic species peaks caused by peptide fragments derived from the target peptide to be analyzed, and judging also that the six group of anionic species peaks selected thereby are a group of anionic species peaks caused by peptide fragments derived from the target peptide to be analyzed, (Step 6) a step identification ion species peaks of peptide fragments per se derived from target peptide to be analyzed, which comprises: with respect to each peak of the sixth group of cationic species peaks, in comparison with the relative intensities of its accompanying cationic species peaks that are relevant to at least one of said relationships (5a-1) to (5a-3) for said peak, selecting peaks whose relative intensities are superior to their accompanying cationic species peaks, and then from the group of the selected peaks, further selecting peaks which is not one of accompanying cationic species peaks that are relevant to at least one of said relationships (5a-1) to (5a-3) for other peak included in said group of the selected peaks and have an inferior relative intensity to other peak, and making up the seventh group of cationic species peaks therewith, meanwhile, with respect to each peak of the sixth group of anionic species peaks, in comparison with the relative intensities of its accompanying anionic species peaks that are relevant to at least one of said relationships (5b-1) to (5b-3) for said peak, selecting peaks whose relative intensities are superior to their accompanying anionic species peaks, and then from the group of the selected peaks, further selecting peaks which is not one of accompanying anionic species peaks that are relevant to at least one of said relationships (5b-1) to (5b-3) for other peak included in said group of the selected peaks and have an inferior relative intensity to other peak, and making up the seventh group of anionic species peaks therewith, (Step 8) a step for identification of group of C-terminal side peptide fragments that are produced from target peptide and a series of reaction products thereof by the treatment for digestion by trypsin, which comprises: with respect to each anionic species peak of the seventh group of anionic species peaks, selecting group of anionic species peaks of which a m/z value difference from the fiducial m/z value of the said anionic species peak is found within the range of less than 200, based on set of the m/z value differences between adjacent peaks that are calculated in the Step 5, from the peaks being present in the fourth group of anionic species peaks, and identifying those anionic species peaks being included in the seventh group of anionic species group, for which there is present, in the thus-selected groups, a peak whose m/z value difference between those peaks is equivalent to the formula weight of natural chain a-amino acid residue: —NH—CH(R)—CO— (R is a side chain of said amino acid residue) or of a-amino acid residue protected by acylation wherein the hydroxy group or amino group of its side chain is modified by substitution with the acyl group used in said N-acylation protection, and then making up the ninth group of anionic species peaks therewith, forming the summed-up group of each anionic species peak of the ninth group of anionic species peaks and each of said anionic species peaks being present in the fourth group of anionic species peaks whose m/z value difference between those peaks has been confirmed in said operation of identification, to be equivalent to the formula weight of amino acid residue, and then defining the group as the tenth group of anionic species peaks, from said largest m/z peak, selecting, in the tenth group of anionic species peaks, an anionic species peak having the largest m/z value, successively identifying, from the tenth group of anionic species peaks, a series of anionic specie peaks each having a m/z value difference between peaks that is equal to the formula weight of amino acid residue, by using, as the finducial point, the m/z value which the anionic species peak with the largest m/z value shows, and then judging that the series of thus-identified peaks as the group consisting of the anionic species peak of C-terminal peptide fragment derived from the original peptide and the anionic species peaks of C-terminal peptides derived from a series of reaction products that are obtained by successive release of C-terminal amino acids of original peptide, which fragments are all produced by the treatment for digestion by trypsin, and (Step 9) a step for assignment of C-terminal amino acid sequence, which comprises: according to a series of said formula weights of amino acid residues that are corresponding to the m/z differences between the anionic species peaks, which have been sequentially assigned in Step 8, based on the identified group consisting of the anionic species peaks of C-terminal peptide fragments that are derived from the original peptide and a series of reaction products resulted from successive release of C-terminal amino acids, which fragments are all produced by the treatment for digestion by trypsin, identifying the sequence of partial amino acids which have been released successively from the C-terminus thereof, wherein the mass spectra used to measure the decreases in molecular weight associated with said successive release of the C-terminal amino acids, are said results of the molecular weight measurements based on the cationic species of (M+H) wherein said process for releasing the C-terminal amino acids successively, as for the sample of the target peptide that has been subjected to separation by gel electrophoresis and is maintained in a state that it is bound on a polyacrylamide gel carrier, comprises the following steps: a step of removing the water solvent impregnated into the polyacrylamide gel carrier by dilution with use of a polar aprotic solvent having no solvency for the polyacrylamide gel substance and having affinity for water, to conduct a dehydration treatment for the polyacrylamide gel carrier, a pretreatment step for the target peptide sample that is still bound on the polyacrylamide gel carrier after carrying out said step for dehydration treatment, in which pretreatment step applying N-acylation protection by the acyl group derived from the alkanoic acid constituting said alkanoic acid anhydride, to the N-terminal amino group of the target peptide with use of a solution of an alkanoic acid anhydride dissolved in a dipolar aprotic solvent that is capable of infiltrating into the polyacrylamide gel substance and keeping it in a swollen state is conducted by immersing, at a temperature selected in a range of 30° C. to 80° C., the polyacrylamide gel carrier in the solution of the alkanoic acid anhydride to allow the alkanoic acid anhydride to act on the target peptide sample that is kept in the bound state; and then removal of said solution is carried out by dilution with use of a polar aprotic solvent having no solvency for the polyacrylamide gel substance and having affinity for the alkanoic acid anhydride as well as the dipolar aprotic solvent, to conduct termination of the N-acylation reaction and removal of the reaction reagent therefor; a step of treatment as for the target peptide sample bound on the polyacrylamide gel carrier, after the pretreatment step of N-acylation protection, comprising steps of immersing, at a temperature selected in a range of 30° C. to 80° C., the polyacrylamide gel carrier in a mixed solution of an alkanoic acid anhydride added with a small amount of a perfluoroalkanoic acid in relative ratio thereto dissolved in a dipolar aprotic solvent that is capable of infiltrating into the polyacrylamide gel substance and keeping it in a swollen state, to allow the alkanoic acid anhydride and in the presence of the small amount of the perfluoroalkanoic acid to act on the target peptide sample being kept in the bound state; thereby, successive release of the C-terminal amino acids results from the reaction process with use of the mixed solution in which formed is a 5-oxazolone-ring structure represented by the following general formula (III): wherein R1 is a side chain of the C-terminal amino acid of the peptide and R2 is a side chain of the amino acid residue positioned just before the C-terminal amino acid, followed by the cleavage of the 5-oxazolone-ring, wherein the formation of the 5-oxazolone structure and the cleavage of the 5-oxazolone ring is carried out in parallel at the same temperature by using the alkanoic acid anhydride in the presence of the small amount of the perfluoroalkanoic acid to successively release the C-terminal amino acids, and removing the mixed solution used in the reaction for successive release of C-terminal amino acids, by dilution with use of a polar aprotic solvent having no solvency for the polyacrylamide gel substance and having affinity for the perfluoroalkanoic acid and the alkanoic acid anhydride as well as the dipolar aprotic solvent, to conduct termination of the releasing reaction and removal of the reaction reagents therefor; and an additional step for hydrolysis treatment and then redehydration treatment, in which step the hydrolysis treatment for said mixture comprising a series of reaction products obtained by the reaction for successive release of C-terminal amino acids is conducted by immersing the polyacrylamide gel carrier in an aqueous solution dissolving a basic nitrogen-containing aromatic compound or a tertiary amine compound therein to allow a water molecule to act, in the presence of said basic nitrogen-containing organic compound, on said peptides of the reaction products being still bound on the polyacrylamide gel carrier, wherein the hydrolysis reaction is carried out by using the water molecule in the presence of the basic, nitrogen-containing, aromatic compound or the tertiary amine compound, whereby the mixture containing the series of reaction products having a carboxyl group at their C-terminal as well as the remains of the original peptide having a carboxyl group at its C-terminus are prepared as the sample to be used for the analysis by means of MALDI-TOF-MS, and then the redehydration treatment for the polyacrylamide gel carrier is performed by removing said aqueous solution infiltrated into the polyacrylamide gel carrier by dilution with use of a polar aprotic solvent having no solvency for the polyacrylamide gel substance and having affinity for water; and wherein said step of measuring the decreases in molecular weight associated with the successive release of the C-terminal amino acids employs a method which comprises: allowing trypsin being soluble in a buffer solution to act on said mixture, after the re-dried up treatment, containing a series of the reaction products finished by hydrolysis treatment to carry out the treatment for the enzymatic digestion specific to trypsin of said peptide chain which holds N-acylation protection as for the N-terminal amino group of the peptide chain as well as to the amino group on the side chain of the lysine residue that may be contained in the peptide chain, and thereby, conducting selective cleavage of the C-terminal side peptide bond of each arginine residue that present in the peptide chain to complete peptide fragmentization, applying a desalting treatment to remove the buffer solution component, followed by recovering and drying the peptide fragments after the digestion treatment by trypsin, followed by drying, next, conducting, as for the dried mixture containing said peptide fragments recovered after the digestion treatment by trypsin, molecular weight measurement for the cationic species of (M+H)