| 摘要 |
This invention discloses systems and methods for determining the sequence of amino acids in a short peptide chain that constructs a protein. The protein is firstly hydrolyzed to various short peptides and amino acid enantiomers. Then, the systems and method are used to separate the short peptides and the amino acid enantiomers, identify qualitatively each of the amino acid enantiomers, and obtain the molecular mass signal for each of the peptides. After that, the identified amino acid enantiomers are used to construct any possible short peptides in an order from the smallest molecular weight dipeptide to higher molecular weight short peptides, and the correct short peptides is confirmed by matching the molecular weight obtained from the mass spectrometry measurement, then, the short peptides are combined to give a large peptide. The process is continued until the whole amino acid sequence of the peptide chain of protein can be determined. |
| 主权项 |
1. A system for determining the amino acid sequence of a protein or a polypeptide, comprising:
a reactor controlled at a predetermined temperature to thermally hydrolyze the polypeptide or the protein to a hydrolyte solution comprising amino acids and their enantiomers, short peptides formed by the amino acids and their enantiomers, and un-hydrolyzed polypeptide or un-hydrolyzed protein; a first liquid chromatography column connecting to an ultraviolet detector so as to separate the amino acids and their enantiomers, short peptides, and un-hydrolyzed polypeptide or un-hydrolyzed protein; a second liquid chromatography column connecting to a fluorescence detector so as to identify the amino acids and their enantiomers; a third liquid chromatography column connecting to a mass spectrometer so as to identify the short peptides and un-hydrolyzed polypeptide or un-hydrolyzed protein and obtain a molecular weight signal (m/z) of mass spectrometry, wherein the mass spectrometer is an ion-trap mass spectrometer with an Electrospray Ionization Interface (ESI), and the first liquid chromatography column, the second liquid chromatography column, and the third liquid chromatography column are initially online parallel connected by a first column switching valve and a second column switching valve, so that the first liquid chromatography column, the second liquid chromatography column, and the third liquid chromatography column are initially run independently; the first column switching valve receiving the separated hydrolyte solution eluted out of the first liquid chromatography column and being switched to connect the first liquid chromatography column and the second liquid chromatography column in series, such that the amino acids and their enantiomers of the hydrolyte solution flow into the second liquid chromatography column for analysis, and the first column switching valve is switched back when the amino acids and their enantiomers completely flow into the second liquid chromatography column; the second column switching valve comprising a sampling loop and being switched when the short peptides of the hydrolyte solution are eluted out from the first liquid chromatography column such that the short peptides of the hydrolyte solution flow into the sampling loop; a third column switching valve connected with the third liquid chromatography column, wherein the second column switching valve and the third column switching valve are simultaneously switched such that the short peptides in the sampling loop flow into the third liquid chromatography column to separate copper ions in a mobile phase for the first liquid chromatography column and the second liquid chromatography column from the short peptides, such that copper ions flow to a waste collector; an injection syringe continually injecting a solvent to the mass spectrometer as the copper ions flowing to the waste collector for a period of time, wherein the third column switching valve is switched after the copper ions are eluted, so that the third liquid chromatography column and the mass spectrometer are connected in series and the short peptides out from the third liquid chromatography column are analyzed by the mass spectrometry for their corresponding m/z; and a computer program to assist the amino acid sequencing procedure by implementing a method comprising the steps of:
constructing one or more possible short peptides from the identified amino acid enantiomers by the second liquid chromatography column; andconfirming the one or more possible short peptides by matching the molecular weight obtained from the molecular weight signal (m/z) of mass spectrometry in an order from small molecular weight to large molecular weight, so as to determine the amino acid sequence of the polypeptide or the protein. |
