Use of microbore high‐performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing
- 1 February 1986
- journal article
- research article
- Published by Wiley in International Journal of Peptide and Protein Research
- Vol. 27 (2), 201-207
- https://doi.org/10.1111/j.1399-3011.1986.tb01811.x
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 (40-60 .mu.L) 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 degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.Keywords
This publication has 17 references indexed in Scilit:
- A microbore high-performance liquid chromatography strategy for the purification of polypeptides for gas-phase sequence analysis. Structural studies on the murine transferrin receptorEuropean Journal of Biochemistry, 1985
- The role of short microbore high-performance liquid chromatography columns for protein separation and trace enrichmentJournal of Chromatography A, 1984
- Polypeptide microsequence analysis with the commercially available gas-phase sequencerAnalytical Biochemistry, 1984
- High-performance liquid chromatography of amino acids, peptides and proteins : XLVIII. Retention behaviour of tryptic peptides of human growth hormone isolated by reversed-phase high-performance liquid chromatography: A comparative study using different chromatographic conditions and predicted elution behaviour based on retention coefficientsJournal of Chromatography A, 1983
- Biomedical applications of derivative spectroscopyTrAC Trends in Analytical Chemistry, 1983
- Similar Amino Acid Sequences: Chance or Common Ancestry?Science, 1981
- Automated amino acid sequence of small peptides utilizing PolybreneAnalytical Biochemistry, 1978
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970
- SURFACE ALLOANTIGENS OF PLASMA CELLSThe Journal of Experimental Medicine, 1970
- A One‐Letter Notation for Amino Acid Sequences*European Journal of Biochemistry, 1968