A microbore high‐performance liquid chromatography strategy for the purification of polypeptides for gas‐phase sequence analysis

Structural studies on the murine transferrin receptor

Boris GREGO, Ian R. VAN DRIEL, Peter A. STEARNE, James W. GODING, Edouard C. NICE, Richard J. SIMPSON

Research output: Contribution to journalArticleResearchpeer-review

30 Citations (Scopus)

Abstract

We describe herein the use of reversed‐phase high‐performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed‐phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20–60 μl). In this manner, purified polypeptides can be loaded directly onto the gas‐phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e. g. lyophilization, evaporation). The application of second‐order‐derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic amino‐acid‐containing peptides in complex tryptic digests is described. N‐terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675–678 (1984); Cell 39, 267–274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.

Original languageEnglish
Pages (from-to)485-491
Number of pages7
JournalEuropean Journal of Biochemistry
Volume148
Issue number3
DOIs
Publication statusPublished - 1 Jan 1985
Externally publishedYes

Cite this

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title = "A microbore high‐performance liquid chromatography strategy for the purification of polypeptides for gas‐phase sequence analysis: Structural studies on the murine transferrin receptor",
abstract = "We describe herein the use of reversed‐phase high‐performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed‐phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90{\%}) in small eluent volumes (20–60 μl). In this manner, purified polypeptides can be loaded directly onto the gas‐phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e. g. lyophilization, evaporation). The application of second‐order‐derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic amino‐acid‐containing peptides in complex tryptic digests is described. N‐terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14{\%} of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675–678 (1984); Cell 39, 267–274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.",
author = "Boris GREGO and {VAN DRIEL}, {Ian R.} and STEARNE, {Peter A.} and GODING, {James W.} and NICE, {Edouard C.} and SIMPSON, {Richard J.}",
year = "1985",
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A microbore high‐performance liquid chromatography strategy for the purification of polypeptides for gas‐phase sequence analysis : Structural studies on the murine transferrin receptor. / GREGO, Boris; VAN DRIEL, Ian R.; STEARNE, Peter A.; GODING, James W.; NICE, Edouard C.; SIMPSON, Richard J.

In: European Journal of Biochemistry, Vol. 148, No. 3, 01.01.1985, p. 485-491.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - A microbore high‐performance liquid chromatography strategy for the purification of polypeptides for gas‐phase sequence analysis

T2 - Structural studies on the murine transferrin receptor

AU - GREGO, Boris

AU - VAN DRIEL, Ian R.

AU - STEARNE, Peter A.

AU - GODING, James W.

AU - NICE, Edouard C.

AU - SIMPSON, Richard J.

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Y1 - 1985/1/1

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AB - We describe herein the use of reversed‐phase high‐performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed‐phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20–60 μl). In this manner, purified polypeptides can be loaded directly onto the gas‐phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e. g. lyophilization, evaporation). The application of second‐order‐derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic amino‐acid‐containing peptides in complex tryptic digests is described. N‐terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675–678 (1984); Cell 39, 267–274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.

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JF - European Journal of Biochemistry

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