QUANTITATIVE STANDARD FOR MASS SPECTROMETRY OF PROTEINS

摘要

The present invention provides a method of determining the absolute amount of a target polypeptide in a sample, said method comprising the following steps: (a) adding (aa) a fusion polypeptide to said sample, said fusion polypeptide comprising (i) at least one tag sequence and (ii) a subsequence of the target polypeptide; and (ab) a known absolute amount of a tag polypeptide comprising or consisting of said tag sequence according to (aa) to said sample, wherein said fusion polypeptide on the one hand is mass-altered as compared to said target polypeptide and said tag polypeptide on the other hand, for example, said fusion polypeptide on the one hand and said target polypeptide and said tag polypeptide on the other hand are differently isotope labeled; (b) performing proteolytic digestion of the mixture obtained in step (a); (c) subjecting the result of proteolytic digestion of step (b), optionally after chromatography, to mass spectrometric analysis; and (d) determining the absolute amount of said target polypeptide from (i) the peak intensities in the mass spectrum acquired in step (c) of said fusion polypeptide, said tag polypeptide and said target polypeptide and (ii) said known absolute amount of said tag polypeptide.