(a) Positive ion mode (ph < 7) Amino acids: ptimization in underivatized formula and fragment patterns in L/ESI-MS analysis R 1 R 2 N + HA R 3 Base Acid R 1 R N H + 2 R 3 + A - Sample [M + H] + (b) Negative ion mode (ph > 7) R H + B R - + H B + Acid Base Sample [M - H] - Yoshinori Takano (JAMSTE) The principles of acid-base theory in solution chemistry for ESI-MS optimization.
(a) Protein AAs (b) Non-protein AAs His, [M+H] + = 156 Met, [M+H] + = 150 Tyr, [M+H] + = 182 Phe, [M+H] + = 166 Arg, [M+H] + = 175 1 Norleu, [M+H] + = 132 Norval, [M+H] + = 118 α-aaa, [M+H] + = 162 Isoval, [M+H] + = 118 α-aba, [M+H] + = 104 Leu, [M+H] + = 132 γ-aba, [M+H] + = 104 Lys, [M+H] + = 147 α-aiba, [M+H] + = 104 Val, [M+H] + = 118 β-aiba, [M+H] + = 104 Glu, [M+H] + = 148 N-Et-Gly, [M+H] + = 104 Pro, [M+H] + = 116 (Phe) β-ala, [M+H] + = 90 Asp, [M+H] + = 134 Sar, [M+H] + = 90 Thr, [M+H] + = 120 Hyp, [M+H] + = 132 Ala, [M+H] + = 90 Time (min) (c) Unhydrolyzed protein AAs Ser, [M+H] + = 106 Gly, [M+H] + = 76 Time (min) Gln, [M+H] + = 147 (Glu) Asn, [M+H] + = 133 (Asp) Time (min)
(a) Protein AAs Gly, [M+H] + = 76 Asp, [M+H] + = 134 H H hemical Formula: 2 H 5 N 2 Exact Mass: 75.03 Molecular Weight: 75.07 [M+H-H 2 ] + H H hemical Formula: 4 H 7 N 4 Exact Mass: 133.04 Molecular Weight: 133.10 80 100 120 140 160 180 200 Ser, [M+H] + = 106 Pro, [M+H] + = 116 [M+H-H 2 ] + H H hemical Formula: 3 H 7 N 3 Exact Mass: 105.04 Molecular Weight: 105.09 HN H hemical Formula: 5 H 9 N 2 Exact Mass: 115.06 Molecular Weight: 115.13 Ala, [M+H] + = 90 H Glu, [M+H] + = 148 H hemical Formula: 3 H 7 N 2 Exact Mass: 89.05 Molecular Weight: 89.09 [M+H-H 2-46] + [M+H-H 2 ] + H hemical Formula: 5 H 9 N 4 Exact Mass: 147.05 Molecular Weight: 147.13 Thr, [M+H] + = 120 Val, [M+H] + = 118 [M+H-H 2 ] + H H H hemical Formula: 4 H 9 N 3 Exact Mass: 119.06 Molecular Weight: 119.12 H H 3 hemical Formula: 5 H 11 N 2 Exact Mass: 117.08 Molecular Weight: 117.15
H Lys, [M+H] + = 147 H Arg, [M+H] + = 175 hemical Formula: 6 H 14 N 2 2 Exact Mass: 146.11 Molecular Weight: 146.19 NH hemical Formula: 6 H 14 N 4 2 Exact Mass: 174.11 Molecular Weight: 174.20 NH H H 3 Leu, [M+H] + = 132 hemical Formula: 6 H 13 N 2 Exact Mass: 131.09 Molecular Weight: 131.17 H Phe, [M+H] + = 166 hemical Formula: 9 H 11 N 2 Exact Mass: 165.08 Molecular Weight: 165.19 H Met, [M+H] + = 150 H Tyr, [M+H] + = 182 S hemical Formula: 5 H 11 N 2 S Exact Mass: 149.05 Molecular Weight: 149.21 H hemical Formula: 9 H 11 N 3 Exact Mass: 181.07 Molecular Weight: 181.19 N H His, [M+H] + = 156 hemical Formula: 6 H 9 N 3 2 Exact Mass: 155.07 Molecular Weight: 155.15 HN H Hyp, [M+H] + = 132 hemical Formula: 5 H 9 N 3 Exact Mass: 131.06 Molecular Weight: 131.13 NH H
(b) Non-protein AAs Asn, [M+H] + = 133 Sar, [M+H] + = 90 H hemical Formula: 4 H 8 N 2 3 Exact Mass: 132.05 Molecular Weight: 132.12 H 3 NH H hemical Formula: 3 H 7 N 2 Exact Mass: 89.05 Molecular Weight: 89.09 Gln, [M+H] + = 147 -Ala, [M+H] + = 90 [M+H-46 -NH 3 ] + H hemical Formula: 5 H 10 N 2 3 Exact Mass: 146.07 Molecular Weight: 146.14 [M+H-H 2 ] + H hemical Formula: 3 H 7 N 2 Exact Mass: 89.05 Molecular Weight: 89.09 N-Et-Gly, [M+H] + = 104 2 H 5 NH H hemical Formula: 4 H 9 N 2 Exact Mass: 103.06 -AiBA, [M+H] + = 104 H 2 H hemical Formula: 4 H 9 N 2 Exact Mass: 103.06
-AiBA, [M+H] + = 104 H -AAA, [M+H] + = 162 H 3 hemical Formula: 4 H 9 N 2 H Exact Mass: 103.06 [M+H-H 2 ] + H hemical Formula: 6 H 11 N 4 Exact Mass: 161.07 Molecular Weight: 161.16 -ABA, [M+H] + = 104 Norval, [M+H] + = 118 H 2 H hemical Formula: 4 H 9 N 2 Exact Mass: 103.06 H hemical Formula: 5 H 11 N 2 Exact Mass: 117.08 Molecular Weight: 117.15 H -ABA, [M+H] + = 104 hemical Formula: 4 H 9 N 2 Exact Mass: 103.06 H Norleu, [M+H] + = 132 hemical Formula: 6 H 13 N 2 Exact Mass: 131.09 Molecular Weight: 131.17 Isoval, [M+H] + = 118 hemical Formula: H 5 H 11N 2 Exact Mass: 117.08 Molecular Weight: 117.15
(a) -H fragment [M + H - 46] + R H H [M + H] + (b) - fragment [M + H - NH 3 ] + H R' H [M + H] + (c) -H fragment H R" H [M + H - H 2 ] + H [M + H] +
Application to L x G//IRMS method: Liquid hromatography x Gas hromatography/ ombustion/ Isotope Ratio Mass Spectrometry R H Amino acid A B D L G IRMS (1D-separation) (2D-separation) magnet ion source A B furnaces m/z 28 29 D N 2 ion source ESI-MS + + + + + Identification and determination Preparative collection on a arbitrary purpose High precision measurement of amino acids for 15 N/ 14 N ompound-specific 15 N/ 14 N analysis Takano et al., IJMS (2015)
References Takano, Y., hikaraishi, Y. and hkouchi, N. (2015) Isolation of underivatized amino acids by ion-pair high performance liquid chromatography for precise measurement of nitrogen isotopic composition of amino acids: development of comprehensive L x G//IRMS method. International Journal of Mass Spectrometry, 379, 16-25. doi: 10.1016/j.ijms.2014.1011.1012. hkouchi, N. and Takano, Y. (2014) rganic nitrogen: sources, fates, and chemistry. Treatise on Geochemistry, Vol. 12, 10: rganic Geochemistry (Edited by Birrer, B., Falkowski, P., Freeman, K.), Elsevier, pp. 251-289. doi: 10.1016/B978-0-08-095975-7.01015-9. hikaraishi Y., Takano Y., gawa. N., and hkouchi, N. (2010) Instrumental optimization for compound-specific nitrogen isotope analysis of amino acids by gas chromatography/combustion/ isotope ratio mass spectrometry. Earth, Life, and Isotopes (edited by N. hkouchi, I. Tayasu, and K. Koba). Kyoto University Press., pp. 367-386. Takano, Y., Kashiyama, Y., gawa,.n., hikaraishi, Y., and hkouchi, N., (2010) Isolation and desalting with cation-exchange chromatography for compound-specific nitrogen isotope analysis of amino acids. Rapid ommunications in Mass Spectrometry, 24, 2317-2323. doi: 10.1002/rcm.4651. Takano, Y., hikaraishi, Y., gawa,. N., Kitazato, H., and hkouchi, N. (2009) ompound-specific nitrogen isotope analysis of D-alanine, L-alanine, and valine: application of diastereomer separation to delta 15 N and microbial peptidoglycan studies. Analytical hemistry, 81, 394-399. doi: 10.1021/ ac802077v.