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 language | English |
|---|---|
| Pages (from-to) | 201-207 |
| Number of pages | 7 |
| Journal | International Journal of Peptide and Protein Research |
| Volume | 27 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 1 Jan 1986 |
| Externally published | Yes |
Keywords
- amino acid
- derivative spectroscopy
- diodearray detector
- gas‐phase sequence analysis
- microbore HPLC
- PC‐1 antigen
- peptide purification
- plasma cell antigen
Cite this
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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. / GREGO, BORIS; DRIEL, IAN R.VAN; GODING, JAMES W.; NICE, EDOUARD C.; SIMPSON, RICHARD J.
In: International Journal of Peptide and Protein Research, Vol. 27, No. 2, 01.01.1986, p. 201-207.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Use of microbore high‐performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing
T2 - Structural studies on the murine plasma cell antigen PC‐1
AU - GREGO, BORIS
AU - DRIEL, IAN R.VAN
AU - GODING, JAMES W.
AU - NICE, EDOUARD C.
AU - SIMPSON, RICHARD J.
PY - 1986/1/1
Y1 - 1986/1/1
N2 - 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.
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.
KW - amino acid
KW - derivative spectroscopy
KW - diodearray detector
KW - gas‐phase sequence analysis
KW - microbore HPLC
KW - PC‐1 antigen
KW - peptide purification
KW - plasma cell antigen
UR - http://www.scopus.com/inward/record.url?scp=0022591364&partnerID=8YFLogxK
U2 - 10.1111/j.1399-3011.1986.tb01811.x
DO - 10.1111/j.1399-3011.1986.tb01811.x
M3 - Article
VL - 27
SP - 201
EP - 207
JO - International Journal of Peptide and Protein Research
JF - International Journal of Peptide and Protein Research
SN - 0367-8377
IS - 2
ER -