Res. Org. Geochem. 31, 1 17 (2015) 地球環境有機分子質量分析マニュアル No.29 技術論文 LC/ESI-MS * ** はじめに Eksborg and Schill, 1973; Gloor and

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1 Res. rg. Geochem. 31, 1 17 (2015) 地球環境有機分子質量分析マニュアル No.29 技術論文 LC/ESI-MS * ** ** ** はじめに Eksborg and Schill, 1973; Gloor and Johnson, 1977; Chaimbault et al., 2000 Knox and Hartwick, 1981; Cecchi, 2008 ElectroSpray Ionization-Mass Spectrometry, ESI-MS; Yamashita and Fenn, 1984 Appendix Liquid Chromatography / Mass Spectrometry, LC/MS Arpino and Guiochon, 1979; Vestal, 1984; Covey et al., ; 2011; Mallet et al., 2004; Antignac et al., 2005; Taylor, 2005 LC / MS Kaneko et al Multiple Reaction Monitoring, MRM femto mol LC / MS GC / MS NIST LC / MS * LC/ESI-MS analysis of underivatized amino acids and mass spectrum ** Yoshinori Takano, Yoshito Chikaraishi, Naohiko hkouchi Department of Biogeochemistry, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka , Japan Corresponding author: Yoshinori Takano takano@jamstec.go.jp Fax:

2 LC / MS 2. 分析方法 2.1. Wako Pure Chemical Industries, Ltd.; Shanghai Hanhong Chemical Co., Ltd.; Sigma-Aldrich Co. LLC. glycine L-α-alanine L-serine L-threonine L-asparagine L-glutamine L-aspartic acid L-glutamic acid L-proline L-valine L-lysine L-leucine L-isoleucine L-methionine L-histidine L-arginine L-phenylalanine L-tyrosine L-hydroxyproline sarcosine β- β-alanine N- N-ethylglycine α- L-α-aminobutyric acid α- α-aminoisobutyric acid β- D,L-β-aminoisobutyric acid γ-γ-aminobutyric acid L-isovaline α- L-α-aminoadipic acid L-norvaline L-norleucine 0.1M HCl ph1 Table HPLC / ESI-MS, Agilent 1100 Takano et al., nmol 160 nmol Hypercarb column mm, particle size 5 µm; Thermo Fisher Scientific Inc mm, particle size 5 µm Cool pocket column cooler, Thermo Fisher Scientific Inc. 10.0A 20 mm Nonafluoropentanoic acid, NFPA; B ml / min 0 B 0 60 B B ml / min Cecchi, 2008 ESI, positive mode m/z L 50 psi 3000V Figure 1 ESI ph M + H + DAD, Agilent nm Appendix 3. 結果と考察 3.1. Figure 2 Figure 3 ph 2

3 LC/ESI-MS Table 1. Summary of the ion-pair reversed-phase LC for underivatized amino acids showing elution order, chemical formula, molecular weight, parent ion, and fragment (m/z) by electrospray ionization mass spectrometry. #1, hydroxyproline is one of the important amino acids in collagen protein. #2, #3, Asparagine (Asn) and glutamine (Gln) will convert to aspartic acid (Asp) and glutamic acid (Glu), respectively, after hydrolysis. #4, The chromatographic co-elution of leucine and isoleucine may occur on this ion-pair LC separation. However, if the eluent and gradient program was modified with same ion-pair reagent, leucine and isoleucine were separated as shown in Takano et al. (2015) and the supplementary information. In the right-hand column, P and NP represent protein amino acid and non-protein amino acid, respectively. Elution order Abbreviation Formula Molecular weight Retention time (min) Parent ions [M+H] + Product ions (m/z) Remarks Glycine Gly C2H5N P Serine Ser C3H7N P Sarcosine Sar C3H7N NP Alanine Ala C3H7N P Hydroxyproline Hyp C5H9N NP (#1) β-alanine β-ala C3H7N NP Threonine Thr C4H9N , 74 P Asparagine Asn C4H8N , 87 P (#2) N-Ethylglycine N-Et-gly C4H9N NP Glutamine Gln C5H10N , 101 P (#3) β-aminoisobutyric acid β-aiba C4H9N NP Aspartic acid Asp C4H7N , 88 P α-aminoisobutyric acid α-aiba C4H9N NP γ-aminobutyric acid γ-aba C4H9N NP Proline Pro C5H9N P α-aminobutyric acid α-aba C4H9N NP Glutamic acid Glu C5H9N , 102 P Isovaline Isoval C5H11N NP Valine Val C5H11N P α-aminoadipic acid α-aaa C6H11N , 116 NP Norvaline Norval C5H11N NP Lysine Lys C6H14N P Leucine Leu C6H13N P Isoleucine Ile C6H13N P Methionine Met C5H11N2S , 104 P Histidine His C6H9N P Norleucine Norleu C6H13N NP Arginine Arg C6H14N P Phenylalanine Phe C9H11N P Tyrosine Tyr C9H11N , 136 P (a) Positive ion mode (ph < 7) R 1 R 2 N + HA R 3 Base Acid (b) Negative ion mode (ph > 7) R 1 R N H + 2 R 3 + A - Sample [M + H] + R C H + B R C - + H B + Acid Base Sample [M - H] - Fig. 1. The principles of acid-base theory in solution chemistry for LC / ESI-MS optimization. ESI-MS M + H + H -CH M + H - HCH + = M + H Figure 3-a M + H + = 118 M + H m/z 72 Figure 4 3

4 Fig. 2. (1/2) (a) Protein AAs Met, [M+H] + = 150 Leu, [M+H] + = 132 Lys, [M+H] + = 147 Val, [M+H] + = 118 Tyr, [M+H] + = 182 Phe, [M+H] + = 166 Arg, [M+H] + = 175 His, [M+H] + = 156 Glu, [M+H] + = 148 Pro, [M+H] + = 116 (Phe) Asp, [M+H] + = 134 Thr, [M+H] + = 120 Hyp, [M+H] + = 132 Ala, [M+H] + = 90 Ser, [M+H] + = 106 Gly, [M+H] + = 76 Time (min) Fig. 2. Extracted ion chromatogram (EIC) of underivatized amino acids on LC / ESI MS analysis. Abbreviations: Glycine, Gly; Serine, Ser; Alanine, Ala; Threonine, Thr; Aspartic acid, Asp; Proline, Pro; Glutamic acid, Glu; Valine, Val; Lysine, Lys; Leucine, Leu; Isoleucine, Ile; Methionine, Met; Histidine, His; Arginine, Arg; Phenylalanine, Phe; Tyrosine, Tyr; Asparagine, Asn; Glutamine, Gln; Sarcosine, Sar; Hydroxyproline, Hyp; β-alanine, β-ala; N-Ethylglycine, N-Et-gly; β-aminoisobutyric acid, β-aiba; α-aminoisobutyric acid, α AiBA; γ-aminobutyric acid, γ-aba; α-aminobutyric acid, α-aba; Isovaline, Isoval; α-aminoadipic acid, α-aaa; Norvaline, Norval; Norleucine, Norleu. A co-injection of threonine and phenylalanine showed the same product ion (m/z 120) at differing retention times. For separation condition, please see Takano et al. (2015). 4

5 LC/ESI-MS Fig. 2. (2/2) (b) Non-protein AAs Figure Norleu, [M+H] + = 132 Norval, [M+H] + = 118 α-aaa, [M+H] + = 162 Isoval, [M+H] + = 118 α-aba, [M+H] + = γ-aba, [M+H] + = 104 α-aiba, [M+H] + = 104 β-aiba, [M+H] + = 104 N-Et-Gly, [M+H] + = 104 β-ala, [M+H] + = 90 Sar, [M+H] + = 90 Time (min) (c) Unhydrolyzed protein AAs Gln, [M+H] + = 147 Asn, [M+H] + = 133 (Asp) (Glu) Time (min) 5

6 (a) -CH fragment R [M + H - 46] + NH 2 CH (b) -NH 2 fragment [M + H - NH 3 ] + R' NH 2 (c) -H fragment H R" [M + H] + H H [M + H] + H NH 2 [M + H] + CH [M + H - H 2 ] + CH Fig. 3. Representative fragmentation patters for diagnosis ion of underivatized amino acids showing, parent ion [M + H] + with (a) CH fragment (-HCH), (b) NH 2 fragment (-NH 3 ), and (c) H fragment (-H 2 ). 2 -NH 2 M + H - NH 3 + = M + H Figure 3-b M + H + = 147 M + H m/z 130 Figure 4 -H M + H - H 2 + = M + H Figure 3-c M + H + = 106 M + H m/z 88 Figure 4 α- β M + H - HCH - H 2 + M + H - HCH - NH 3 + M + H + = 120 M + H + = 166 M + H = 120 EIC, m/z 120 Figure 2 2 Table 1 6

7 LC/ESI-MS Fig. 4. (a) (1/5) (a) Protein AAs Gly, [M+H] + = 76 H H Chemical Formula: C 2 H 5 N 2 Exact Mass: Molecular Weight: Ser, [M+H] + = 106 [M+H-H 2 ] + H Chemical Formula: C 3 H 7 N 3 Exact Mass: Molecular Weight: H Ala, [M+H] + = 90 H Chemical Formula: C 3 H 7 N 2 Exact Mass: Molecular Weight: Thr, [M+H] + = 120 [M+H-H 2 ] + CH H H Chemical Formula: C 4 H 9 N 3 Exact Mass: Molecular Weight: m/z Fig. 4. (a) Underivatized protein amino acids and (b) non-protein amino acids for the identification. bserved product ions and their fragmentation patterns by ESI MS were shown. The chemical formula, exact mass, molecular weight, and theoretical values of their corresponding masses (m/z) are also shown. 7

8 Fig. 4. (a) (2/5) Asp, [M+H] + = 134 H [M+H-H 2 ] + C H Chemical Formula: C 4 H 7 N 4 Exact Mass: Molecular Weight: Pro, [M+H] + = 116 HN C H Chemical Formula: C 5 H 9 N 2 Exact Mass: Molecular Weight: H Glu, [M+H] + = 148 [M+H-H 2-46] + [M+H-H 2 ] + Chemical Formula: C 5 H 9 N 4 Exact Mass: Molecular Weight: C H Val, [M+H] + = 118 H H 3 Chemical Formula: C 5 H 11 N 2 Exact Mass: Molecular Weight: m/z 8

9 LC/ESI-MS Fig. 4. (a) (3/5) H Lys, [M+H] + = 147 [M+H-NH 3 ] + Chemical Formula: C 6 H 14 N 2 2 Exact Mass: Molecular Weight: NH 2 Leu, [M+H] + = 132 H H 3 Chemical Formula: C 6 H 13 N 2 Exact Mass: Molecular Weight: NH 2 H Met, [M+H] + = 150 [M+H-NH 3 ] + S Chemical Formula: C 5 H 11 N 2 S Exact Mass: Molecular Weight: H His, [M+H] + = 156 N Chemical Formula: C 6 H 9 N 3 2 Exact Mass: Molecular Weight: NH m/z 9

10 Fig. 4. (a) (4/5) H Arg, [M+H] + = 175 NH Chemical Formula: C 6 H 14 N 4 2 Exact Mass: Molecular Weight: C NH NH 2 H Phe, [M+H] + = 166 Chemical Formula: C 9 H 11 N 2 Exact Mass: Molecular Weight: H Tyr, [M+H] + = 182 [M+H-NH 3 ] + Chemical Formula: C 9 H 11 N 3 Exact Mass: Molecular Weight: H Hyp, [M+H] + = 132 HN C H Chemical Formula: C 5 H 9 N 3 Exact Mass: Molecular Weight: H m/z 10

11 LC/ESI-MS Fig. 4. (a) (5/5) Asn, [M+H] + = 133 [M+H-NH 3 ] + C NH 2 H Chemical Formula: C 4 H 8 N 2 3 Exact Mass: Molecular Weight: m/z: (100.0%), (4.5%) [M+H-NH 3 ] + Gln, [M+H] + = 147 H [M+H-46 -NH 3 ] + C NH 2 Chemical Formula: C 5 H 10 N 2 3 Exact Mass: Molecular Weight: m/z: (100.0%), (6.3%) Table 1 Corona CAD, Charged Aerosol Detector Evaporative Light Scattering Detector, ELSD Corona CAD Appendix: Thermo Fisher Scientific Inc. 4. 今後の展望 LC 2 2 Hamase et al D / L N- ; Chikaraishi et al., 2010 LC x GC LC-GC Chikaraishi et al., Chikaraishi et al., 2010; Takano et al., 2010,

12 Fig. 4. (b) (1/3) (b) Non-protein AAs Sar, [M+H] + = 90 H 3 C NH C H Chemical Formula: C 3 H 7 N 2 Exact Mass: Molecular Weight: β-ala, [M+H] + = 90 [M+H-H 2 ] + C H Chemical Formula: C 3 H 7 N 2 Exact Mass: Molecular Weight: N-Et-Gly, [M+H] + = 104 C 2 H 5 NH C H Chemical Formula: C 4 H 9 N 2 Exact Mass: Molecular Weight: β-aiba, [M+H] + = 104 H 2 C H Chemical Formula: C 4 H 9 N 2 Exact Mass: Molecular Weight: Fig. 4. (a) Underivatized protein amino acids and (b) non-protein amino acids for the identification. bserved product ions and their fragmentation patterns by ESI MS were shown. The chemical formula, exact mass, molecular weight, and theoretical values of their corresponding masses (m/z) are also shown. 12

13 LC/ESI-MS Fig. 4. (b) (2/3) α-aiba, [M+H] + = 104 Chemical Formula: C 4 H 9 N 2 C C H Exact Mass: Molecular Weight: γ-aba, [M+H] + = 104 H 2 C C H Chemical Formula: C 4 H 9 N 2 Exact Mass: Molecular Weight: α-aba, [M+H] + = 104 H Chemical Formula: C 4 H 9 N 2 Exact Mass: Molecular Weight: Isoval, [M+H] + = 118 C C H Chemical Formula: C 5 H 11N 2 Exact Mass: Molecular Weight:

14 Fig. 4. (b) (3/3) H α-aaa, [M+H] + = 162 [M+H-H 2 ] + C H Chemical Formula: C 6 H 11 N 4 Exact Mass: Molecular Weight: Norval, [M+H] + = 118 H Chemical Formula: C 5 H 11 N 2 Exact Mass: Molecular Weight: Norleu, [M+H] + = 132 H Chemical Formula: C 6 H 13 N 2 Exact Mass: Molecular Weight: m/z Takano et al., 2009; hkouchi and Takano, 2014 Chikaraishi and hkouchi, 2010 gawa et al., 2010 δ 13 C Itahashi et al., 2014 謝辞 MEXT No ; Y.T. JST CREST; N.., Y.C. 14

15 LC/ESI-MS 引用文献 Antignac J.-P., de Wasch K., Monteau F., De Brabander H., Andre F. and Le Bizec B. (2005) The ion suppression phenomenon in liquid chromatography mass spectrometry and its consequences in the field of residue analysis. Anal. Chim. Acta 529, Arpino P. J. and Guiochon G. (1979) LC / MS coupling. Anal. Chem. 51, Cecchi T. (2008) Ion pairing chromatography. Critical Rev. Anal. Chem. 38, Chaimbault P., Petritis K., Elfakir C. and Dreux M. (2000) Ion-pair chromatography on a porous graphitic carbon stationary phase for the analysis of twenty underivatized protein amino acids. J. Chromatogr. A 870, Chikaraishi Y., gawa.n., Kashiyama Y., Takano Y., Suga H., Tomitani A., Miyashita H., Kitazato H. and hkouchi, N. (2009) Determination of aquatic food-web structure based on compound-specific nitrogen isotopic composition of amino acids. Limnol. ceanogr.: Methods 7, Chikaraishi 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 hkouchi N., Tayasu I. and Koba K.), Kyoto Univ Press, pp Chikaraishi Y. and hkouchi N. (2010) An improved method for precise determination of carbon isotopic composition of amino acids. Earth, Life, and Isotopes (Edited by hkouchi N., Tayasu I. and Koba K.), Kyoto Univ Press, pp N- - GC / MS Res. rg. Geochem. 25, Covey T.R., Lee E.D., Bruins A.P. and Henion J.D. (1986) Liquid chromatography / mass spectrometry. Anal. Chem. 58, 1451A-1461A. Eksborg S. and Schill G. (1973) Ion pair partition chromatography of organic ammonium compounds. Anal. Chem. 45, Gloor R. and Johnson E.L. (1977) Practical aspects of reverse phase ion pair chromatography. J. Chromatogr. Sci. 15, Hamase K., Nakauchi Y., Miyoshi Y., Koga R., Kusano N., nigahara H., Naraoka H., Mita H., Kadota Y. and Nishio Y. (2014) Enantioselective determination of extraterrestrial amino acids using a two-dimensional chiral high-performance liquid chromatographic system. Chromatogr. 35, Itahashi Y., Takano Y., gawa. N., hkouchi N. and Yoneda M. (2014) Radiocarbon dating of single amino acid in archaeological bone specimens. Anthropological Sci., 122, 190. Kaneko M., Takano Y., Chikaraishi Y., gawa N.., Asakawa S., Watanabe K., Shima S., Krueger M., Matsushita M., Kimura H. and hkouchi N. (2014) Quantitative analysis of coenzyme F430 in environmental samples: a new diagnostic tool for methanogenesis and anaerobic methane oxidation. Anal. Chem. 86, Knox J.H. and Hartwick R.A. (1981) Mechanism of ion-pair liquid chromatography of amines, neutrals, zwitterions and acids using anionic hetaerons. J. Chromatogr. A. 204, Mallet C.R., Lu Z. and Mazzeo J.R. (2004) A study of ion suppression effects in electrospray ionization from mobile phase additives and solid phase extracts. Rapid Comm. Mass Spectrom. 18, LC / MS, LC / MS / MS pp pp 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 gawa.n., Nagata T., Kitazato H. and hkouchi N. (2009) Ultra sensitive elemental analyzer / isotope ratio mass spectrometer for stable nitrogen and carbon isotope analyses. Earth, Life, and Isotopes 15

16 (Edited by hkouchi N., Tayasu I. and Koba K.), Kyoto Univ Press, pp LC / MS. pp Takano Y., Chikaraishi Y., gawa.n., Kitazato H. and hkouchi N. (2009) Compound-specific nitrogen isotope analysis of D-, L-alanine and valine: application of diastereomer separation to δ 15 N and microbial peptidoglycan studies. Anal. Chem. 81, Takano Y., Kashiyama Y., gawa.n., Chikaraishi Y. and hkouchi N. (2010) Isolation and desalting with cation-exchange chromatography for compound-specific nitrogen isotope analysis of amino acids. Rapid Comm. Mass Spectrom. 24, Takano Y., Chikaraishi 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 LC x GC / C / IRMS method. Int. J. Mass Spectrom. 379, Taylor P.J. (2005) Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography electrospray tandem mass spectrometry. Clinical Biochem. 38, Vestal M.L. (1984) High-performance liquid chromatography-mass spectrometry. Science 226, GC / MS Res. rg. Geochem. 25, Yamashita M. and Fenn J.B. (1984) Electrospray ion source. Another variation on the free-jet theme. J. Phys. Chem. 88, Abbreviations : LC / MS, Liquid Chromatography / Mass Spectrometry; ESI-MS, ElectroSpray Ionization-Mass Spectrometry; EIC, Extracted Ion Chromatogram; DAD, Diode Array Detector; MRM, Multiple Reaction Monitoring; NIST, National Institute of Standards and Technology; Corona CAD, Corona Charged Aerosol Detector; ELSD, Evaporative Light Scattering Detector. 16

LC/ESI-MS Nebulizer (gas shown in red) Electrospray ions Solvent spray MS Neutral Molecules Clusters Salts appendix. 1. Illustration of typical electrospray source (ESI).

17 LC/ESI-MS Nebulizer (gas shown in red) Electrospray ions Solvent spray MS Neutral Molecules Clusters Salts appendix. 1. Illustration of typical electrospray source (ESI). Arg (i) Corona CAD Phe Tyr (ii) UV 260 nm Intensity (DAD) Intensity (CAD) (iii) multi-absorbance Wavelength (nm) Time (min) appendix. 2. Comparison of responses determined by other online detectors for corona CAD (Thermo Fisher Scientific Inc.) and the diode array detector (DAD) with the UV absorbance. Modified after Takano et al. (2015) 17

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Res. Org. Geochem. 31, 1 17 (2015) 地球 環境有機分子質量分析マニュアル No.29 技術論文 LC/ESI-MS * ** ** ** はじめに Eksborg and Schill, 1973; Gloor and

Res. Org. Geochem. 31, 1 17 (2015) 地球環境有機分子質量分析マニュアル No.29 技術論文 LC/ESI-MS * ** はじめに Eksborg and Schill, 1973; Gloor and