A Novel UPLC-MS/MS Method Identifies Organ-Specific Dipeptide Profiles

Abstract

Background: Amino acids have a central role in cell metabolism, and intracellular changes contribute to the pathogenesis of various diseases, while the role and specific organ distribution of dipeptides is largely unknown.

Method: We established a sensitive, rapid and reliable UPLC-MS/MS method for quantification of 36 dipeptides. Dipeptide patterns were analyzed in brown and white adipose tissues, brain, eye, heart, kidney, liver, lung, muscle, sciatic nerve, pancreas, spleen and thymus, serum and urine of C57BL/6N wildtype mice and related to the corresponding amino acid profiles.

Results: A total of 30 out of the 36 investigated dipeptides were detected with organ-specific distribution patterns. Carnosine and anserine were most abundant in all organs, with the highest concentrations in muscles. In liver, Asp-Gln and Ala-Gln concentrations were high, in the spleen and thymus, Glu-Ser and Gly-Asp. In serum, dipeptide concentrations were several magnitudes lower than in organ tissues. In all organs, dipeptides with C-terminal proline (Gly-Pro and Leu-Pro) were present at higher concentrations than dipeptides with N-terminal proline (Pro-Gly and Pro-Leu). Organ-specific amino acid profiles were related to the dipeptide profile with several amino acid concentrations being related to the isomeric form of the dipeptides. Aspartate, histidine, proline and serine tissue concentrations correlated with dipeptide concentrations, when the amino acids were present at the C- but not at the N-terminus.

Conclusion: Our multi-dipeptide quantification approach demonstrates organ-specific dipeptide distribution. This method allows us to understand more about the dipeptide metabolism in disease or in healthy state.

Keywords: UPLC; biofluids; dipeptides; mass spectrometry; metabolism; tissue.

Figure 1

Chromatograms of 36 dipeptides and the internal standard norleucine.0.5 pmol per analyte were loaded on column. (A) Dipeptides with higher signal intensity; (B) those with lower intensity yields. Compounds are corresponding to the numbers in the chromatograms as follows: 1. Ser-His; 2. Gly-His; 3. His-Ser; 4. Carnosine; 5. Ser-Gln; 6. Ala-His; 7. Anserine; 8. Gly-Asp; 9. Asp-Gln; 10. Glu-Ser; 11. Gly-Glu; 12. His-Ala; 13. Ala-Gln; 14. Ala-Gly; 15. Ser-Ala; 16. Glu-Glu; 17. Pro-Gly; 18. Gly-Sar; 19. Ala-Glu; 20. Ala-Ala; 21. Gly-Pro; 22. γ-Glu-ε-Lys; 23. Ala-Tyr; 24. Ala-Pro; 25. Leu-His; 26. Tyr-Ala; 27. His-Leu; 28. Arg-Phe; 29. Gly-Phe; 30. Val-Tyr; 31. Pro-Leu; 32. Ala-Phe; 33. Phe-Ala; 34. Norleucine (internal standard); 35. Aspartame; 36. Tyr-Phe; 37. Leu-Pro.

Figure 2

Distribution of dipeptides in murine tissues and biofluids. (a) Heat map showing the concentrations for 28 dipeptides and (b) carnosine and anserine in murine tissues (n = 6, except for urine n = 5). Mean values are given in fmol/mg tissue. In serum, the concentrations are given in fmol/µL and in urine in fmol/mg creatinine.

Figure 3

The sum of 28 dipeptides from various tissues and biofluids (as depicted in Figure 2). Sums are presented as violin blot showing median (bold line) with standard deviation (n = 6; except for urine n = 5). Concentrations are given in fmol/mg tissue in tissue and in fmol/µL in tissue and in fmol/mg creatinine in urine.

Figure 4

Organ-specific distribution of the six most abundant dipeptides (excluding anserine and carnosine). Box plots demonstrating the organ-specific distribution of the dipeptides Ala-Ala, Ala-Gln, Asp-Gln, Glu-Ser, Gly-Asp, His-Ser, showing median as bold line with standard deviation and ● indicating outliers (n = 6). The two glutamine dipeptides (Ala-Gln and Asp-Gln) and Ala-Ala were the major dipeptides in liver, Glu-Ser in muscle and spleen, Gly-Asp in spleen and thymus and His-Ser in muscle.

Figure 5

Distribution of the six most abundant dipeptides (except carnosine and anserine) in urine and serum. Box plot showing the distribution of Ala-Gln, Gly-Asp, Glu-Ser, His-Ser, Ala-Ala and Asp-Gln in urine in fmol/mg creatinine (n = 5) and serum in fmol/µL (n = 6), showing median as bold line with standard deviation and ● indicating outliers.

Figure 6

Distribution of four stereoisomeric dipeptides in murine tissue. Box plot showing the organ-specific concentration of four structural isomers (Ala-Phe/Phe-Ala; His-Ser/Ser-His; Pro-Leu/Leu-Pro; Gly-Pro/Pro-Gly), showing median as a bold line with standard deviation and ● indicating outliers (n = 6).

Figure 7

Distribution of amino acids in murine tissues and biofluids. Heat map showing the concentrations for 17 amino acids concentration (n = 6) in murine tissues (adipose tissue, brain, eyes, heart, kidney, liver, lungs, muscle, nervus ischiadicus, pancreas, spleen and thymus) and biofluids (serum, urine). Mean values are given in pmol/mg tissue (n = 6). In serum, the concentrations are given in pmol/µL and in urine in pmol/mg creatinine (n = 5).

Figure 8

Correlation of dipeptides and respective amino acids in murine tissues and biofluids.