Mass spectrometric analysis of rabbit and bovine trypsin-solubilized cytochrome b5.
The sequence and blocking group of the amino-terminal 15 amino acids of rabbit trypsin-solubilized cytochrome b5 were determined by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). The molecular weights of peptides generated from a Staphylococcus aureus V8 protease digest of this protein were determined by LSIMS analysis and the two peptides containing the blocked amino-terminus were sequenced by tandem mass spectrometry to yield the sequence; N-acetyl-Ala-Ala-Glu-Ser-Asp-Lys-Asp-Val-Lys-Tyr-Tyr-Thr-Leu-Glu-Glu. Comparison of this sequence with a recently reported cDNA sequence (Dariush et al., 1988) indicates that Gln at position 3 is selectively deamidated, although no other discrepancies were found. Intact rabbit and bovine trypsin-solubilized cytochrome b5 were also analyzed by LSIMS on a high-field mass spectrometer equipped with a diode array detector. Mass measurement of the unresolved protonated molecular ion peak tops gave average molecular weights of 9462.2 +/- 2 and 9502.3 +/- 2 for bovine and rabbit trypsin-solubilized cytochrome b5, respectively. In both cases, these molecular weights correspond to a cytochrome b5 fragment consisting of amino acids Asp(7)-Arg(88). The average molecular weight for the rabbit amino-terminal-blocked form of trypsin-solubilized cytochrome b5 was found to be 10,144.5 +/- 2, which was consistent with the molecular weight predicted for the extended N-acetylated form (residues 1-88) of Mr 10,146.1.
Gibson
B W
BW
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
Falick
A M
AM
Lipka
J J
JJ
Waskell
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D015786:Cytochromes b5 / Q000032:analysis* / Q000302:isolation & purification; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis*; D011817:Rabbits; D012697:Serine Endopeptidases; D014357:Trypsin
D010446:Peptide Fragments; D015786:Cytochromes b5; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin