Mass spectrometric analysis of the structure of gamma II bovine lens crystallin.
The amino acid sequence of bovine gamma II-crystallin has been verified by a combination of electrospray and fast atom bombardment mass spectrometry. The molecular weight of gamma II, isolated by gel filtration and ion exchange chromatography, was determined to be 20,967 +/- 3 by electrospray mass spectrometry. Another aliquot of gamma II was completely digested by trypsin in a medium of 20% CH3CN and 0.1 M Tris, pH 8.2. The tryptic peptides were separated by reversed phase HPLC and identified by their molecular weights, as determined by fast atom bombardment mass spectrometry (FABMS). The identification of each peptide was confirmed by digesting the peptide further to give new peptides whose molecular weights were also determined by FABMS and related to the proposed amino acid sequences. The data from both types of mass spectrometric analyses were consistent with the sequence previously proposed by Hay et al. (J. Biol. Chem. 1987, 146, 332-338), including threonine at position 119. The FAB mass spectrum of one HPLC fraction suggested that disulfide bonding between Cys 18 and Cys 22 was present in at least half the protein preparation. Whether the Cys 18/Cys 22 disulfide bond was present in native gamma II or was produced during isolation or enzymic digestion could not be determined from these studies. Samples that had been stored for several weeks showed that several of the cysteines had become disulfide bonded. These studies illustrate the power of mass spectrometric techniques to accurately confirm the primary structure of proteins and to identify post-translational modifications.
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Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, IN 47907.
Smith
J B
JB
Smith
D L
DL
Edmonds
C G
CG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016339:Spectrometry, Mass, Fast Atom Bombardment
D003459:Crystallins