Sequential digestion of polypeptides for mass spectrometric analysis
AssigneeAmgen Inc.
InventorsDa REN, Melissa SATO
Life of this patent
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Abstract
The disclosed methods are directed to preparing polypeptides for multi-attribute analysis. The polypeptides are optionally denatured, reduced, and/or alkylated before being subjected to a first digestion. Following the first digestion the large and small fragments resulting from the digestion are separated from each other. A second digestion is then performed on the larger of the fragments. All of the fragments from the two digestions are then analyzed chromatographically, electrophoretically, or spectrometrically, or a combination of these methods. The methods are especially useful for the preparation of therapeutic polypeptides for analysis, especially those that are not easily cleaved.
Claims12
-24. (canceled)
A method of preparing a polypeptide for analysis, comprising:
- a. denaturing, reducing, and alkylating the polypeptide;
- b. digesting the polypeptide with trypsin to produce large trypsin-cleaved polypeptide fragments and small trypsin-cleaved polypeptide fragments;
- c. separating the large and small trypsin-cleaved polypeptide fragments into a first aliquot and a second aliquot;
- d. digesting the large trypsin-cleaved polypeptide fragments of the first aliquot with neutrophil elastase;
- e. combining the first aliquot and second aliquot at about a 1:1 ratio;
- and
- f. analyzing the combined aliquots of step (e).
Dependencies are inferred from the claim text.