| 摘要 |
There is provided a method of determining the absolute amount of at least one target protein in a sample, said method comprising the following steps: (a) adding an absolute amount of a mass-altered standard protein for each target protein to said sample; (b) proteolytically cleaving target protein and standard protein from step (a) using at least one protease to obtain cleaved peptides; (c) subjecting cleaved peptides from step (b), optionally after chromatography, to mass spectrometry (MS) to obtain a spectrum, which MS comprises filtering using an isolation window of 1 -3 Th for co-isolation of ions from each pair of target protein and standard protein; and (d) determining the absolute amount of said target protein(s) in said sample from the spectrum obtained in step (c) and said absolute amount(s) of corresponding standard protein(s) added in step (a), wherein (i) each standard protein comprises a target sequence that is identical to a subsequence of its target protein, (ii) the target sequence comprises at least one cleavage amino acid residue at which the at least one protease cleaves, (iii) the mass of each cleavage amino acid residue in the target sequence of the standard protein is altered by 1 -5 Da, such as 1 -4 Da or 1 -2 Da, with respect to the mass of the corresponding cleavage amino acid residue(s) in the subsequence of the target protein and (iv) the amino acid residues in the target sequence of the standard protein other than the cleavage amino acid residue(s) are not mass altered with respect to the corresponding amino acid residues in the subsequence of the target protein. An isotopically labelled fusion protein that can be used in the method is also provided. |
