Mapping of recombinant hemoglobin using immobilized trypsin cartridges.
A tryptic mapping procedure has been developed for a recombinant hemoglobin (rHb1.1) using an immobilized trypsin cartridge. Apohemoglobin is passed through the trypsin cartridge and the products of the digestion are captured directly onto an in-line C18 reversed-phase column. The peptides are then separated using a gradient elution. This new procedure is rapid and reproducible and can be fully automated. The total time of analysis is less than 2 h. The mapping of apohemoglobins produced an unexpected isomerization of two peptides: beta8,9 (K66-K82) and alpha8,9 (K61-K90). It appears that the isomerization may occur through transpeptidation followed by proteolysis at a newly generated site next to the site of ligation. This mapping procedure can be a useful tool for research and routine analysis of proteins.
Lippincott
J
J
Somatogen, Inc., 2545 Central Avenue, Boulder, Colorado 80301, USA.
Hess
E
E
Apostol
I
I
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized; D006454:Hemoglobins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry*; D018629:Spectrometry, Mass, Secondary Ion; D013696:Temperature; D014357:Trypsin
D001059:Apoproteins; D004800:Enzymes, Immobilized; D006454:Hemoglobins; D011994:Recombinant Proteins; C023478:apohemoglobin; D014357:Trypsin