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Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier (Polybrene), and high pressure liquid chromatography
Abstract
We have combined the use of a nonprotein carrier (Polybrene), high pressure liquid chromatography, and modifications in Edman chemistry with the improvements of a commercial spinning cup sequenator suggested by Wittmann-Liebold [Wittmann-Liebold, B. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 1415] to analyze amino acid phenylthiohydantoins obtained from automated Edman degradation of microquantities of polypeptide directly without the use of radiolabel. This approach has allowed us to determine the sequence of the N-terminal 47 residues of sperm whale myoglobin starting with 200 pmol of protein, 77 residues of an antibody light chain with 5 nmole of protein, and 54 residues of an antibody heavy chain with 8 nmol of protein. In addition, we completely sequenced a hydrophobic 14-residue peptide at the 1.5-nmol level. Our technique of direct analysis for microsamples is capable of providing routine, extende N-terminal sequence analysis for nanomole and subnanomole levels of polypeptides and protines, and it also is applicable to analysis of more classical sample quantities.
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Cited by
Dynorphin-(1-13), an extraordinarily potent opioid peptide.
Goldstein, A., Tachibana, S., Lowney, L. I., Hunkapiller, M., & Hood, L. (1979) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.76.12.6666 · Cited by 882
Self-citation[36] High-sensitivity sequencing with a gas-phase sequenator
Hunkapiller, M. W., Hewick, R. M., Dreyer, W. J., & Hood, L. E. (1983) · Methods in Enzymology · doi:10.1016/s0076-6879(83)91038-8 · Cited by 659
Self-citationPrimary structure of corticotropin-releasing factor from ovine hypothalamus
Spiess, J., Rivier, J., Rivier, C., & Vale, W. (1981) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.78.10.6517 · Cited by 424
MacGillivray, R. T., Mendez, E., Shewale, J. G., Sinha, S. K., Lineback-Zins, J., & Brew, K. (1983) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)32696-6 · Cited by 275
Metadata
- Authors
- Hunkapiller, M. W., & Hood, L. E.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1978-05-30
- Volume
- 17
- Issue
- 11
- Pages
- 2124-2133
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2016266875
- Cited by
- 383
- References
- 0
- FWCI
- 45.74
- Type
- journal article
- Language
- en
- Open access
- closed
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- Hunkapiller, M. W., & Hood, L. E. (1978). Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier (Polybrene), and high pressure liquid chromatography. Biochemistry. https://doi.org/10.1021/bi00604a016
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