Analysis of enzymatically released peptides by reverse-phase high performance liquid chromatography from picomole amounts of apolipoproteins separated on polyacrylamide isoelectric focusing gels

L. V. Pereira, P. J. Dolphin

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Résumé

Methods were developed for the peptide analysis of individual isoproteins of human apolipoproteins separated on urea-polyacrylamide isoelectric focusing (IEF) gels. After IEF the proteins were fixed in the gel matrix by trichloroacetic acid precipitation. Low molecular weight contaminants, including ampholytes, were removed and the proteins were chemically desialylated. Enzymatic digestions with L-1-tosyl-2-phenylethylchloromethyl ketone - trypsin, chymotrypsin, or with thermolysin were accomplished within the gel matrix. The proteolytically released peptides were analyzed by reverse-phase high performance liquid chromatography. These methods facilitated the comprehensive analysis of protein structural differences between individual isoproteins of apolipoproteins in duplicate with as little as 1-2 nmol of each isoprotein, without the use of radiolabels. Human apolipoproteins A-I, C and E were analysed by these methods.

Langue d'origineEnglish
Pages (de-à)790-797
Nombre de pages8
JournalCanadian Journal of Biochemistry
Volume60
Numéro de publication8
DOI
Statut de publicationPublished - 1982

ASJC Scopus Subject Areas

  • General Medicine

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Pereira, L. V., & Dolphin, P. J. (1982). Analysis of enzymatically released peptides by reverse-phase high performance liquid chromatography from picomole amounts of apolipoproteins separated on polyacrylamide isoelectric focusing gels. Canadian Journal of Biochemistry, 60(8), 790-797. https://doi.org/10.1139/o82-098