Multiple sequential fraction collection of peptides and glycopeptides by high-performance capillary electrophoresis.
Multiple sequential fraction collection of peptides and glycopeptides by high-performance capillary electrophoresis (HPCE) under applied voltage has been demonstrated from complex tryptic peptide maps. The collection methodology was adapted from a high-resolution glycopeptide mapping procedure and, as such, requires active temperature control of the sample, electrophoresis vials, and collections vials because the electrophoresis buffer system is higher conductive. Resolution was compromised in the preparative HPCE separation due to heavy sample loading and to reduced voltage. The latter was a requirement for this buffer system in order to control Joule heating at the current levels employed; collections were routinely performed at approximately 1.5 W/m. The collection buffer was optimized by the addition of 12% methanol (v/v), thereby improving collection yields. Tryptic non-glycopeptides were group collected; secondary analysis of the HPCE collections agreed with analytical separations with respect to the number of peptides contained in a given fraction. Sequentially collected peptide fractions were analyzed by Edman sequencing and MALDI mass spectrometry to verify peptide identity and sequence. Consistent peptide sequence or mass measurements were obtained for repeat collections. The isolation of the single pure glycopeptide indicates that unique glycopeptide structures can be collected by HPCE and then analyzed by other methods.
Boss
H J
HJ
Amgen Inc., Thousand Oaks, California 91320-1789, USA.
Rohde
M F
MF
Rush
R S
RS
D000595:Amino Acid Sequence; D005591:Chemical Fractionation; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000302:isolation & purification*; D006020:Glycopeptides / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006020:Glycopeptides; D010455:Peptides; D011994:Recombinant Proteins; D004921:Erythropoietin