Use of microbore high‐performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing

Structural studies on the murine plasma cell antigen PC‐1

BORIS GREGO, IAN R.VAN DRIEL, JAMES W. GODING, EDOUARD C. NICE, RICHARD J. SIMPSON

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Abstract

A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed‐phase high performance liquid chromatography on short (10 cm or less) microbore (1–2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC‐1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed‐phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (> 90%) and in small eluent volumes (40–60μL) which can be loaded directly onto the gas‐phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode‐array detector for identifying tryptophan‐containing peptides from on‐the‐fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan‐containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.

Original languageEnglish
Pages (from-to)201-207
Number of pages7
JournalInternational Journal of Peptide and Protein Research
Volume27
Issue number2
DOIs
Publication statusPublished - 1 Jan 1986
Externally publishedYes

Keywords

  • amino acid
  • derivative spectroscopy
  • diodearray detector
  • gas‐phase sequence analysis
  • microbore HPLC
  • PC‐1 antigen
  • peptide purification
  • plasma cell antigen

Cite this

@article{702884c8358c4b968d59b63f44bcd1a0,
title = "Use of microbore high‐performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing: Structural studies on the murine plasma cell antigen PC‐1",
abstract = "A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed‐phase high performance liquid chromatography on short (10 cm or less) microbore (1–2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC‐1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed‐phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (> 90{\%}) and in small eluent volumes (40–60μL) which can be loaded directly onto the gas‐phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode‐array detector for identifying tryptophan‐containing peptides from on‐the‐fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan‐containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11{\%} of the molecule.",
keywords = "amino acid, derivative spectroscopy, diodearray detector, gas‐phase sequence analysis, microbore HPLC, PC‐1 antigen, peptide purification, plasma cell antigen",
author = "BORIS GREGO and DRIEL, {IAN R.VAN} and GODING, {JAMES W.} and NICE, {EDOUARD C.} and SIMPSON, {RICHARD J.}",
year = "1986",
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language = "English",
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AU - DRIEL, IAN R.VAN

AU - GODING, JAMES W.

AU - NICE, EDOUARD C.

AU - SIMPSON, RICHARD J.

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AB - A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed‐phase high performance liquid chromatography on short (10 cm or less) microbore (1–2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC‐1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed‐phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (> 90%) and in small eluent volumes (40–60μL) which can be loaded directly onto the gas‐phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode‐array detector for identifying tryptophan‐containing peptides from on‐the‐fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan‐containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.

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KW - microbore HPLC

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KW - peptide purification

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