Mass spectrometry of lens crystallins: bovine beta-crystallins.
The bovine beta-crystallins have been isolated by gel permeation chromatography (GPC) and fast protein liquid chromatography (FPLC). Electrospray ionization mass spectrometric examination of the resulting fractions confirms the relative molecular masses of three bovine beta-crystallins, i.e. beta B1, beta B2 and beta A2, only one of which (beta B2) has been reported previously by mass spectrometry. The sequence of beta B3 is shown to be incorrect as the identity of this protein was confirmed by FPLC, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass analysis of a tryptic digest of this protein. ESI analysis of the remaining beta subunits, i.e. beta A1, beta A3 and beta A4, suggests the published sequences of these proteins also contain errors. The major components observed in the beta H (i.e. octameric) and beta L (trimeric) aggregates of the beta-crystallins are shown to be consistent with subunit compositions determined by SDS-PAGE. A number of cleavage products of beta-crystallins were also identified, i.e. species with masses corresponding to beta B1(2)-203, beta B1(2)-197, beta B8(2)-204, beta B1(2)-196 were detected in the ESI mass spectra following isolation of the beta B2 fraction from one-year-old lenses by FPLC. A species with mass of beta B6(1)-244 is also observed in the ESI mass spectrum of the beta H fraction isolated by GPC. The largest and most hydrophobic of the beta-crystallins, beta B1, is detected in the beta H aggregate but not following FPLC purification of a fraction containing beta B1 and beta B2. A variety of different methods (including MALDI) were used to examine this fraction but none enabled the successful analysis of beta B1 in the deaggregated protein. Finally, this work demonstrates the advantages and some of the difficulties in mass spectrometric characterization of this class of proteins.
Kilby
G W
GW
Department of Chemistry, University of Wollongong, NSW, Australia.
Truscott
R J
RJ
Stuchbury
G M
GM
Sheil
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007908:Lens, Crystalline / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D003459:Crystallins; D010455:Peptides; D014357:Trypsin