Amino Acid Analysis, Using Postcolumn Ninhydrin Detection, in a Biotechnology Laboratory

  • Frank D. Macchi
  • Felicity J. Shen
  • Rodney G. Keck
  • Reed J. Harris
Part of the Methods in Molecular Biology™ book series (MIMB, volume 159)


Although lacking the speed and sensitivity of more widely heralded techniques such as mass spectrometry, amino acid analysis remains an indispensable tool in a complete biotechnology laboratory responsible for the analysis of protein pharmaceuticals.


Amino Acid Analysis Peptide Fraction Methionine Sulfoxide Lithium Citrate Standard Chromatogram 
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  1. 1.
    Moore S. and Stein W. H. (1958) Chromatographic determination of amino acids by the use of automatic recording equipment. Methods Enzymol. 6, 819–831.CrossRefGoogle Scholar
  2. 2.
    Schuster R. (1988) Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivatization and high performance liquid chromatography. J. Chromatog. 431, 217–284.Google Scholar
  3. 3.
    Heinrickson R. L. and Meredith S. C. (1983) Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal. Biochem. 136, 65–74.CrossRefGoogle Scholar
  4. 4.
    van Wandlen C. and Cohen S. A. (1997) Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxysuc-cinimidyl carbamate-derivatized amino acid mixtures. J. Chromatog. A 763, 11–22.CrossRefGoogle Scholar
  5. 5.
    Kisumi M., Sugiura M., and Chibata I. (1976) Biosynthesis of norvaline, norleu-cine and homoisoleucine in Serratia marcescens. J. Biochem. 80, 333–339.PubMedGoogle Scholar
  6. 6.
    Tsai L. B., Lu H. S., Kenney W. C., Curless C. C., Klein M. L., Lai P.-H., et al. (1988) Control of misincorporation of de novo synthesized norleucine into recom-binant interleukin-2 in E. coli. Biochem. Biophys. Res. Commun. 156, 733–739.CrossRefGoogle Scholar
  7. 7.
    Bogosian G., Violand B. N., Dorward-King E. J., Workman W. E., Jung P. E., and Kane J. F. (1989) Biosynthesis and incorporation into protein of norleucine by Escherichia coli. J. Biol. Chem. 264, 531–539.PubMedGoogle Scholar
  8. 8.
    Kivirikko K. I., Myllyla R., and Pihlajaniemi T. (1992) Hydroxylation of proline and lysine residues in collagens and other animal and plant proteins, in Posttrans-lational Modifications of Proteins (Harding J. J. and Crabbe M. J., eds.), CRC, Boca Raton, FL, pp. 1–51.Google Scholar
  9. 9.
    Molony M. S., Wu S.-L., Keyt L., and Harris R. J. (1995) The unexpected presence of hydroxylysine in non-collagenous proteins, in Techniques in Protein Chemistry VI (Crabbe J., ed.), Academic, San Diego, CA, pp. 91–98.CrossRefGoogle Scholar
  10. 10.
    Kornfeld R. and Kornfeld S. (1985) Assembly of asparagine-linked oligosaccha-rides. Annu. Rev. Biochem. 54, 631–664.PubMedCrossRefGoogle Scholar
  11. 11.
    Tarentino A. L., Gomez C. M., and Plummer T. H. (1985) Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry 24, 4665–4671.PubMedCrossRefGoogle Scholar
  12. 12.
    O’Connell B., Tabak L. A., and Ramasubbu N. (1991) The influence of flanking sequences on O-glycosylation. Biochem. Biophys. Res. Commun. 180, 1024–1030.CrossRefGoogle Scholar
  13. 13.
    Wilson I. B. H., Gavel Y., and von Heijne G. (1991) Amino acid distributions around O-linked glycosylation sites. Biochem. J. 275, 528–534.Google Scholar
  14. 14.
    Pisano A., Packer N. H., Redmond J. W., Williams K. L., and Gooley A. A. (1994) Characterization of O-linked glycosylation motifs in the glycopeptide domain of bovine ?-casein. Glycobiology 4, 837–844.PubMedCrossRefGoogle Scholar
  15. 15.
    Garnick R. L., Solli N. J., and Papa P. A. (1988) The role of quality control in biotechnology: an analytical perspective. Anal. Chem. 60, 2546–2557.PubMedCrossRefGoogle Scholar
  16. 16.
    Lundell N. and Schreitmüller T. (1999) Sample preparation for peptide mapping — a pharmaceutical quality-control perspective. Anal. Biochem. 266, 31–47.PubMedCrossRefGoogle Scholar
  17. 17.
    Jones M. D., Merewether L. A., Clogston C. L., and Lu H. S. (1994) Peptide map analysis of recombinant human granulocyte stimulating factor: elimination of me-thionine modification and nonspecific cleavages. Anal. Biochem. 216, 135–146.PubMedCrossRefGoogle Scholar
  18. 18.
    Allen G. (1989) Determination of the carboxy-terminal residue, in Sequencing of Proteins and Peptides, Elsevier, Amsterdam and New York, pp. 67–71.Google Scholar
  19. 19.
    Grunau J. A. and Swaider J. M. (1992) Chromatography of 99 amino acids and other ninhydrin-reactive compounds in the Pickering lithium gradient system. J. Chromatog. 594, 165–171.CrossRefGoogle Scholar
  20. 20.
    Clarke A. P., Jandik P., Rocklin R. D., Liu Y., and Avdalovic N. (1999) An integrated amperometry waveform for the direct, sensitive detection of amino acids and amino sugars following anion-exchange chromatography. Anal. Chem. 71, 2774–2781.CrossRefGoogle Scholar
  21. 21.
    Strydom D. J. (1996) Amino acid analysis using various carbamate reagents for precolumn derivatization, in Techniques in Protein Chemistry VII (Marshak D. R., ed.), Academic, San Diego, CA, pp. 331–339.CrossRefGoogle Scholar
  22. 22.
    Molony M. S., Quan C., Mulkerrin M. G., and Harris R. J. (1998) Hydroxylation of Lys residues reduces their susceptibility to digestion by trypsin and lysyl en-dopeptidase. Anal. Biochem. 258, 136–137.PubMedCrossRefGoogle Scholar
  23. 23.
    Harris R. J. and Spellman M. W. (1993) O-Linked fucose and other post-transla-tional modifications unique to EGF modules. Glycobiol. 3, 219–224CrossRefGoogle Scholar
  24. 24.
    Yan S. B., Chao Y. B., and van Halbeek H. (1993) Novel Asn-linked oligosaccha-rides terminating in GalNAcβ(1 →4)[Fucα(1 →3)]GlcNAcβ(1 →●) are present in recombinant human Protein C expressed in human kidney 293 cells. Glycobiology 3, 597–608.PubMedCrossRefGoogle Scholar
  25. 25.
    Chan A. L., Morris H. R., Panico M., Eteinne A. T., Rogers M. E., Gaffney P., et al. (1991) A novel sialylated N-acetylgalactosamine-containing oligosaccharide is the major complex-type structure present in Bowes melanoma tissue plasminogen activator. Glycobiology 1, 173–185.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Frank D. Macchi
    • 1
  • Felicity J. Shen
    • 1
  • Rodney G. Keck
    • 1
  • Reed J. Harris
    • 1
  1. 1.Analytical Chemistry DepartmentGenentech Inc.San Francisco

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