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Mass Spectrometry of Lens Crystallins: Bovine β‐Crystallins
Author(s) -
Kilby Greg W.,
Truscott Roger J. W.,
Stuchbury Glen M.,
Sheil Margaret M.
Publication year - 1996
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19960115)10:1<123::aid-rcm440>3.0.co;2-p
Subject(s) - chemistry , fast protein liquid chromatography , chromatography , mass spectrometry , crystallin , electrospray ionization , molecular mass , beta (programming language) , gel permeation chromatography , gel electrophoresis , high performance liquid chromatography , biochemistry , enzyme , organic chemistry , polymer , computer science , programming language
The bovine β‐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 β‐crystallins, i.e. βB 1 , βB 2 and βA 2 , only one of which (βB 2 ) has been reported previously by mass spectrometry. The sequence of βB 3 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 β subunits, i.e. βA 1 , βA 3 and βA 4 , suggests the published sequences of these proteins also contain errors. The major components observed in the β H (i.e. octameric) and β L (trimeric) aggregates of the β‐crystallins are shown to be consistent with subunit compositions determined by SDS‐PAGE. A number of cleavage products of β‐crystallins were also identified, i.e. species with masses corresponding to βB 1–203 2 , βB 1–197 2 , βB 8–204 2 , βB 1–196 2were detected in the ESI mass spectra following isolation of the βB 2fraction from one‐year‐old lenses by FPLC. A species with mass of βB 6–244 1is also observed in the ESI mass spectrum of the β Hfraction isolated by GPC. The largest and most hydrophobic of the β‐crystallins, βB 1 , is detected in the β Haggregate but not following FPLC purification of a fraction containing βB 1and βB 2 . A variety of different methods (including MALDI) were used to examine this fraction but none enabled the successful analysis of βB 1in the deaggregated protein. Finally, this work demonstrates the advantages and some of the difficulties in mass spectrometric characterization of this class of proteins.