Isolation of carboxyl-termini and blocked amino-termini of viral proteins by high-performance cation-exchange chromatography.
The strong cation-exchanger, PolySulfoethyl Aspartamide, has been assessed as a medium for isolation of carboxyl-terminal and blocked amino-terminal peptides from tryptic digests of small quantities of viral proteins. Peptides with a single positive charge, the blocked amino-terminal peptides of ovalbumin and the Newcastle disease virus (NDV) matrix protein and carboxyl-terminal peptides of ovalbumin and the NDV nucleocapsid protein, eluted in early ion-exchange fractions and were readily isolated in homogeneous form by subsequent reversed-phase HPLC. Some early ion-exchange fractions also contained singly charged peptides derived by "chymotryptic-like" cleavage, whilst other peptides eluted in these fractions due to their highly acidic character. Terminal sequences with additional basic residues were isolated from later eluting ion-exchange fractions. Peptides with this property included the blocked amino-terminus of the NDV nucleocapsid protein and a portion of the carboxyl-terminus of the NDV matrix protein. Hitherto undescribed polymorphism in the amino-terminal region of ovalbumin was revealed in this study. Truncated peptides from the carboxyl-terminus of the NDV matrix protein were also detected. The presence of these peptides could be a reflection of carboxyl-terminal processing of the matrix protein. The strategy described herein should be of general utility for selective microisolation of carboxyl-terminal peptides and blocked amino-terminal peptides from tryptic digests of proteins.
Gorman
J J
JJ
Commonwealth Scientific and Industrial Research Organization, Australian Animal Health Laboratory, Geelong, Vic.
Shiell
B J
BJ
D000596:Amino Acids / Q000032:analysis; D002213:Capsid / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D009522:Newcastle disease virus / Q000737:chemistry; D010047:Ovalbumin / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014357:Trypsin; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification*
D000596:Amino Acids; D010455:Peptides; D014764:Viral Proteins; D010047:Ovalbumin; D014357:Trypsin