Detecting leucine and its isomers using differential esterification mass spectrometry
Methods of evaluating molecular isomers of branched-chain amino acids are featured. The methods can include: derivatizing one or more molecular isomers of branched-chain amino acids in a sample comprising a branched-chain amino acid labeled with one or more heavy atoms as a first standard; adding, to the sample, after derivatization, a nonderivatized or derivatized branched chain amino acid that is labeled with one or more heavy atoms, as a second standard; evaluating the sample using tandem mass spectrometry; and detecting peaks indicative of derivatized and nonderivatized forms of one or more branched-chain amino acids in the sample.
1. A method of detecting a presence or absence of leucine or an isomer thereof in a sample, the method comprising:
providing a test sample;
adding to the test sample a first standard comprising a first amino acid that is labeled with one or more heavy atoms, thereby providing a modified test sample, wherein the first amino acid is leucine or an isomer thereof;
subjecting the modified test sample to esterification conditions, thereby providing an esterified sample;
adding to the esterified sample a second standard comprising an esterified form or a non-esterified form of a second amino acid that is labeled with one or more heavy atoms, thereby providing a modified esterified sample, wherein the second amino acid is leucine or an isomer thereof;
evaluating the modified esterified sample using tandem mass spectrometry; and
detecting signals indicative of esterified and non-esterified forms of at least two different amino acids in the modified esterified sample, wherein the at least two different amino acids are selected from leucine and isomers of leucine, thereby distinguishing between the at least two amino acids and detecting the presence or absence of leucine or an isomer thereof in the test sample.
2. The method of claim 1 , wherein the esterification conditions provide an esterified form of the first standard, wherein the second standard comprises the esterified form of the second amino acid, and wherein the esterified form of the first standard and the second standard have different molecular weights as a result of different heavy atom labeling.
3. The method of claim 1 , wherein the second standard comprises the non-esterified form of the second amino acid, and wherein the first standard prior to the esterification conditions and the second standard have different molecular weights as a result of different heavy atom labeling.
4. The method of claim 1 , further comprising determining an esterification efficiency of leucine or an isomer thereof by reference to the first standard and the second standard.
5. The method of claim 4 , further comprising determining a concentration of leucine or an isomer thereof in the test sample using the esterification efficiency.
6. The method of claim 1 , wherein the at least two different amino acids include one at least two of leucine, isoleucine, and allo-isoleucine.
7. The method of claim 1 , wherein the esterification conditions are carried out with an alcohol.
8. The method of claim 7 , wherein the alcohol is selected from the group consisting of butanol, isopropyl alcohol, ethanol, and methanol.
9. The method of claim 1 , wherein during the esterification conditions, less than 100% of the first amino acid is esterified.
10. The method of claim 1 , wherein the first standard is selected from the group consisting of labeled leucine, labeled isoleucine, and labeled allo-isoleucine.
11. The method of claim 1 , wherein the second standard is selected from the group consisting of labeled leucine, labeled isoleucine, and labeled allo-isoleucine.
12. The method of claim 1 , wherein the detecting signals comprises acquiring data signals representing butylated and unbutylated forms of leucine or an isomer thereof.
13. A method of diagnosing a metabolic disorder, the method comprising:
providing a test sample from a subject;
adding to the test sample a first standard comprising a first amino acid that is labeled with one or more heavy atoms, thereby providing a modified test sample, wherein the first amino acid is leucine or an isomer thereof;
subjecting the modified test sample to esterification conditions, thereby providing an esterified sample;
adding to the esterified sample a second standard comprising an esterified form or a non-esterified form of a second amino acid that is labeled with one or more heavy atoms, thereby providing a modified esterified sample, wherein the second amino acid is leucine or an isomer thereof;
evaluating the modified esterified sample using tandem mass spectrometry; and
detecting signals indicative of esterified and non-esterified forms of at least two different amino acids in the modified esterified sample, wherein the at least two different amino acids are selected from leucine and isomers of leucine, thereby distinguishing between the at least two amino acids and determining a level of leucine or an isomer thereof in the test sample;
wherein the level of the leucine or the isomer thereof in the test sample being altered relative to a level of leucine or isomer thereof in one or more reference samples is an indication that the subject has, or is at risk of developing, a metabolic disorder characterized by an altered level of the leucine or the isomer thereof.
14. The method of claim 13 , wherein the metabolic disorder is Maple Syrup Urine Disorder (MSUD).
15. The method of claim 13 , wherein the subject is a human.
16. The method of claim 13 , wherein the subject is a newborn human.
17. The method of claim 13 , further comprising determining an esterification efficiency of leucine or an isomer thereof by reference to the first standard and the second standard.
18. The method of claim 17 , further comprising determining a concentration of leucine or an isomer thereof in the test sample using the esterification efficiency.
19. The method of claim 13 , wherein the at least two different amino acids include at least two of leucine, isoleucine, and allo-isoleucine.
20. The method of claim 13 , wherein the esterification conditions provide an esterified form of the first standard, wherein the second standard comprises the esterified form of the second amino acid, and wherein the esterified form of the first standard and the second standard have different molecular weights as a result of different heavy atom labeling.
21. The method of claim 13 , wherein the second standard comprises the non-esterified form of the second amino acid, and wherein the first standard prior to the esterification conditions and the second standard have different molecular weights as a result of different heavy atom labeling.
22. The method of claim 13 , wherein the esterification conditions are carried out with an alcohol.
23. The method of claim 22 , wherein the alcohol is selected from the group consisting of butanol, isopropyl alcohol, ethanol, and methanol.
24. The method of claim 13 , wherein during the esterification conditions, less than 100% of the first amino acid is esterified.
25. The method of claim 13 , wherein the first standard is selected from the group consisting of labeled leucine, labeled isoleucine, and labeled allo-isoleucine.
26. The method of claim 13 , wherein the second standard is selected from the group consisting of labeled leucine, labeled isoleucine, and labeled allo-isoleucine.