title: Identification of amino acid epimerization and isomerization in crystallin proteins by tandem LC-MS.
abstract: Post-translational modifications that do not result in a change in mass are particularly difficult to detect by mass spectrometry. For example, isomerization of aspartic acid or epimerization of any chiral residue within a peptide do not lead to mass shifts but can be identified by examination of independently acquired tandem mass spectra or by combination with another technique. For analysis of a biological sample, this means that liquid chromatography or some other type of separation must be used to first separate the isomers from one another. Furthermore, each specific m/z of interest must be sampled repeatedly to allow for comparison of the tandem mass spectra from each separated isomer, which contrasts with the traditional approach in proteomics where the goal is typically to avoid resampling the same m/z. We illustrate that isomerization and epimerization of peptides can be identified in this fashion by examination of long-lived crystallin proteins extracted from a sheep eye lens. Tandem mass spectrometry relying on a combination of radical directed dissociation (RDD) and collision induced dissociation (CID) following separation by liquid chromatography was used to identify modified peptides. Numerous sites of isomerization and epimerization, including several that have not been previously identified, were determined with peptide specificity. It is demonstrated that the specific sites of amino acid isomerization within each peptide can be identified by comparison with synthetic peptides. For α-crystallin proteins, the sites that undergo the greatest degree of isomerization correspond to disordered regions, which may have important implications on chaperone functionality within the context of aging.
authors.affiliation: Department of Chemistry, University of California , Riverside, California 92521, United States.
authors.forename: Yuanqi
authors.initials: Y
authors.lastname: Tao
authors.forename: Ryan R
authors.initials: RR
authors.lastname: Julian
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D007536:Isomerism; D007908:Lens, Crystalline / Q000737:chemistry; D010455:Peptides / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational; D012756:Sheep; D053719:Tandem Mass Spectrometry; D038201:alpha-Crystallins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D010455:Peptides; D038201:alpha-Crystallins