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.

Racemization of Serine Residues Catalyzed by Dihydrogen Phosphate Ion: A Computational Study

“…It seems that an H 2 PO 4 − ion has an effective proton relay ability. It has recently been elucidated that the majority of Ser (and Asp) residues that are susceptible to racemization reside in flexible regions of proteins [26][27][28]. If a Ser residue can adopt such a conformation as shown in Figure 2 and then its α-proton and carbonyl oxygen are exposed to solvent water, it is likely to undergo enolization (and hence racemization) by the mechanism revealed in the present study.…”

“…Indeed, we did not find a corresponding stable conformation for the main chain of the analogous alanine (Ala)-containing compound (Ace-Ala-Nme); instead, a conformer with ϕ = 73 • and ψ = −56 • was located because of the formation of an intramolecular hydrogen bond between the oxygen of Ace and the hydrogen of Nme (1.932 Å). It has been experimentally observed that Ser residue racemization occurs mainly in flexible regions of proteins [26][27][28]. Therefore, the existence of the above reactive conformer unique to Ser may be one of the reasons why Ser residues are prone to racemization.…”

“…Racemization of Ser residues has also been observed for crystallin proteins from sheep, pig, and cow eye lenses, especially in structurally disordered N-terminal and C-terminal regions [27,28]. An age-dependent increase in D-Ser residues was also demonstrated for proteins in articular cartilage and tooth dentin [29,30].…”

Isomerization of aspartyl residues in crystallins and its influence upon cataract

“…Recently, many outstanding methods for the analysis of peptide-containing isomers [25,26] and epimers [27][28][29][30][31] using MS2 spectra have been reported. Although these methods are excellent, however, they require advanced instruments and expertise for analysis.…”

Problems and Pitfalls in the Analysis of Amygdalin and Its Epimer

“…4,6,9−13,15−18 As a result, it is not surprising that many recent publications on amygdalin analysis do not mention/identify the S-amygdalin, which is always present to some extent depending on the storage conditions, extraction solvents, and analytical procedures. Although in a few specific cases, using advanced instrumentation such as MS-MS that may allow differentiation of epimers, 19,20 ideally it is desired and usually necessary that the epimers are separated chromatographically. Thus, the separation of the natural and its unwanted isomerized amygdalin is best done chromatographically.…”