EXPRESSION QUANTIFICATION USING MASS SPECTROMETRY
In various aspects, the present teachings provide systems, methods, assays and kits for the absolute quantitation of protein expression. In various aspects, the present teachings provide methods of determining the concentration of about the top forty-one proteins present in human plasma. In various aspects, the present teachings provide methods of determining the absolute concentration of one or more proteins using standard samples of signature protein fragments and parent-daughter ion transition monitoring (PDITM). In various embodiments, the absolute concentration of multiple isoforms of a biomolecule in a sample, multiple proteins in a biological process, a combination of multiple samples, or combinations thereof, can be determined in a multiplex fashion using the present teachings. In various aspects, provided are methods of assessing the state of a biological system including, but not limited to, the disease state of an animal.
1 . A method for assessing the biological state of a sample, comprising the steps of:
providing a standard sample comprising a signature peptide for each protein of interest;
selecting a diagnostic daughter ion for each signature peptide;
labeling the one or more proteins of interest in two or more samples of interest with different chemical moieties for each sample, the two or more samples of interest thereby being differentially labeled;
labeling one or more standard samples with a chemical moiety;
combining, to produce a combined sample, at least a portion of the one or more labeled standard samples with at least a portion of two or more differentially labeled samples, the differentially labeled samples being labeled with a different chemical moiety than the one or more labeled standard samples combined therewith;
loading at least a portion of the combined sample on a chromatographic column;
subjecting at least a portion of the eluent from the chromatographic column to multiple reaction monitoring, the transmitted parent ion m/z range of each multiple reaction monitoring scan including a m/z value of one or more of the labeled signature peptides and the transmitted daughter ion m/z range of each multiple reaction monitoring scan including a m/z value one or more of the selected diagnostic daughter ions corresponding to the transmitted labeled signature peptide;
measuring the ion signal of one or more of the selected diagnostic daughter ions using said multiple reaction monitoring; and
determining the concentration of a protein of interest in one or more of the two or more samples of interest based at least on a comparison of the measured ion signal of a selected diagnostic daughter ion corresponding to the protein of interest from a sample of interest to the measured ion signal for the selected diagnostic daughter ion corresponding to the protein of interest from a labeled standard sample; and
assessing the biological state of the sample based at least on a comparison of the relative concentrations of two or more proteins in one or more of the two or more samples to the concentration of two or more corresponding proteins in one or more of the standard samples; wherein
the one or more proteins of interest are one or more of the proteins listed in column 1 of Table 4; and further wherein
the signature peptide comprises one or more of the peptides listed in column 3 of Table 4, which corresponds to a respective protein of interest listed in column 1 of Table 4.
2 . The method of claim 1 , wherein the step of labeling proteins of interest in one or more standard samples comprises labeling proteins of interest with an isotopically coded affinity tag, and wherein the step of labeling proteins of interest in different samples comprises labeling proteins of interest with an isotopically coded affinity tag.
3 . The method of claim 1 , wherein the step of labeling proteins of interest in one or more standard samples comprises labeling proteins of interest with an isobaric tag, and wherein the step of labeling proteins of interest in different samples comprises labeling proteins of interest with an isobaric tag.
4 . The method of claim 1 , wherein the step of labeling proteins of interest in one or more standard samples comprises labeling proteins of interest with a mass differential tag, and wherein the step of labeling proteins of interest in different samples comprises labeling proteins of interest with a mass differential tag.
5 . The method of claim 1 , wherein the one or more standard samples comprise a pooled reference sample.
6 . The method of claim 1 , further comprising the step of subjecting at least a portion of the combined sample to digestion to produce a digested combined sample prior to loading of at least a portion of the combined sample on a chromatographic column, and wherein the portion of the combined sample on a chromatographic column is all or a portion of the digested combined sample.
7 . The method of claim 6 , wherein the digestion comprises chemical digestion.
8 . The method of claim 6 , wherein the digestion comprises enzymatic digestion.
9 . The method of claim 1 , wherein one or more of the one or more of the standard samples are subjected to a digestion prior to being combined with the two or more labeled samples of interest to produce a combined sample.
10 . The method of claim 9 , wherein the digestion comprises chemical digestion.
11 . The method of claim 9 , wherein the digestion comprises enzymatic digestion.
12 . The method of claim 1 , wherein the step of determining the concentration of a protein of interest comprises determining the absolute concentration of the protein of interest.
13 . The method of claim 1 , the step of determining the concentration of a protein of interest comprises determining the relative concentration of the protein of interest, wherein the labeled standard sample comprises a pooled reference sample.
14 . The method of claim 1 , wherein the step of assessing the biological state of a sample comprises a comparison based at least on a comparison of the absolute concentrations of two or more proteins in one or more of the two or more samples to the concentration of two or more corresponding proteins in one or more of the standard samples.
15 . The method of claim 1 , wherein the biological state comprises one or more of a disease state, a response to a chemical agent, or combinations thereof.
16 . The method of claim 1 , wherein:
the transmitted parent ion m/z range of each multiple reaction monitoring scan for a protein of interest listed in column 1 of Table 4 includes an m/z value listed in column 6 of Table 4, which corresponds to a respective signature peptide listed in column 3 of Table 4, which corresponds to a respective protein of interest listed in column 1 of Table 4; and
the transmitted diagnostic daughter ion m/z range of each multiple reaction monitoring scan for a protein of interest listed in column 1 of Table 4 includes an m/z value listed in column 7 of Table 4, which corresponds to a respective parent ion m/z value listed in column 6 of Table 4, which corresponds to a respective signature peptide listed in column 3 of Table 4, which corresponds to a respective protein of interest listed in column 1 of Table 4.