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US 11,442,067 B2Granted
Peptide mapping method for sequence identification of insulin and insulin analogues
AssigneeBiocon Limited
InventorsPhanichand Kodali, Krishnappa Mane, Srivatsa Koduru, Ashutosh Sudhir Naik, Laxmi Adhikary
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Abstract
The invention relates to peptide mass fingerprinting technique for the proteins such as Human insulin and insulin analogs. The insulin analogues can vary at least by one amino acid, which is elusive to distinguish by currently available analytical methods. The invention further allows sequence confirmation of the peptide wherein the run time of the method is forty minutes. This method could be applied for molecules up to 50 kDa.
Claims8
A method for determining the amino acid sequence and mass of a polypeptide or mixture of polypeptide(s), comprising the steps of:
- a) preparing polypeptide sample by dissolving the polypeptide in HCl and adjusting pH by Tris;
- b) digesting the polypeptide sample by addition of endoproteinase Glu C in ratio of 25:1;
- c) reducing the digested sample of step (b) by addition of 1M dithiothreitol;
- d) performing HPLC analysis followed by mass spectrometry analysis of the polypeptide of step (c);
- e) comparing the molecular mass of the polypeptide with the molecular mass of a corresponding known polypeptide, thereby determining the identity of the polypeptide;
- wherein organic solvent used is in step (d) is a non-salt buffer and the method allows sequence confirmation of multiple fragments at the same time.
3 more elements
Dependencies are inferred from the claim text.