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.
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 (greater than 90%) and in small eluent volumes (40-60 microL) 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.
D000595:Amino Acid Sequence; D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte; D000954:Antigens, Surface / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007519:Isoantigens / Q000302:isolation & purification*; D051379:Mice; D010446:Peptide Fragments / Q000302:isolation & purification; D014357:Trypsin
D000944:Antigens, Differentiation, B-Lymphocyte; D000954:Antigens, Surface; D007519:Isoantigens; D010446:Peptide Fragments; D014357:Trypsin