Isomerization Of Aspartyl Residues Research Articles

IsoAsp-Quest: Workflow development for isoAsp identification using database searches.

A recent study reported that isomerization of aspartyl residues (Asp) occurs in various tissues and proteins in vivo. For a comprehensive analysis of post-translational modifications, the MS-based proteomic approach is a straightforward method; however, the isomerization of Asp does not alter its molecular weight. Therefore, a unique method is required to analyze Asp isomers using mass spectrometry. Herein, we present a novel strategy, isoAsp-Quest, which is a database search-oriented isoAsp identification method. isoAsp is specifically converted to 18O-labeled Lα-Asp by the enzymatic reaction of protein L-isoaspartyl-O-methyltransferase (PIMT) in 18O water with a mass shift of 2 Da, which, in principle, enables us to distinguish Asp isomers. However, in practice, a labeled Lα-Asp signal overlaps with that of endogenous Lα-Asp, making detection challenging. Therefore, degradation of the endogenous Lα-Asp peptide by AspN and subsequent removal of AspN were performed prior to the PIMT reaction. This strategy was applied to bovine lens α-crystallin. Consequently, several Asp isomerization sites, consistent with human αA-crystallin, were identified in bovine αA-crystallin, indicating that this strategy is also effective for biological proteins. Therefore, isoAsp-Quest enables the analysis of Lβ-Asp in a straightforward and rapid workflow, which may be useful for the quality control of protein products and biomarker discovery.

Presence of aspartic dipeptides with β peptide bond and/or D-aspartyl residue in rat blood after ingestion of porcine liver protein hydrolysate

Background: Animal experiments have demonstrated that oral administration of a porcine liver protein hydrolysate (LPH) ameliorates alcohol-induced liver toxicity, as well as exercise- and concanavalin A-induced low locomotor activity in mice. The peptides responsible for the beneficial effect have not yet been identified. Recently, presence of food-derived peptides in human blood has been detected post ingestion of other food protein hydrolysates. These peptides are prolyl, hydroxyprolyl, or pyroglutamyl di- and tri-peptides, and resist exopeptidase digestion. Some of these peptides exert significant biological roles in vitro and in vivo, which have been associated with the biological response post ingestion. The objective of the present study was to identify the food-derived peptides in rat blood after ingestion of LPH.Results: In the in vitro exopeptidase digest of LPH, pyroglutamyl, prolyl, hydroxyprolyl, and aspartic dipeptides were identified. The aspartic peptides (Asp-Val, Asp-Ile, Asp-Leu, and Asp-Phe) showed multiple peaks by LC-MS/MS, indicating the presence of isomers. Four isomers with L- and D-aspartyl residues, and α and β peptide bonds were present in each sequence. After administration of LPH, the amounts of unusual aspartic dipeptides with β peptide bond and/or D-aspartyl residue significantly increased in rat plasma, while those of the other usual aspartic peptides did not.Conclusions: Racemization and isomerization of aspartyl residues in peptides occur during the preparation of LPH or following its digestion. The unusual aspartic peptides have a potential for carrying out diverse biological activities.Key words: Peptide, food protein hydrolysate, aspartic, isopeptide, bioavailability, D-amino acid

Isomerization of aspartyl residues in crystallins and its influence upon cataract

BackgroundAge-related cataracts, which probably form due to insolubilization of lens proteins, can lead to loss of vision. Although the exact reason is unknown, lens protein aggregation may be triggered by increases in PTMs such as d-β-, l-β- and d-α-Asp isomers. These isomers have been observed in aged lens; however, there have been few quantitative and site-specific studies owing to the lack of a quick and precise method for distinguishing between d- and l-Asp in a peptide or protein. Scope of reviewWe describe a new method for detecting peptides containing Asp isomers at individual sites in any protein by using an LC–MS/MS system combined with commercial enzymes that specifically react with different isomers. We also summarize current data on the effect of Asp isomerization on lens crystallins. Major conclusionsThe new technique enabled the analysis of isomers of Asp residues in lens proteins precisely and quickly. An extensive proportion of Asp isomerization was observed at all Asp sites of crystallins in the insoluble fraction of aged lens. In addition, d-amino acid substitutions in crystallin-mimic peptides showed altered structural formation and function. These results indicate that isomerization of Asp residues affects the stability, structure and inter-subunit interaction of lens crystallins, which will induce crystallin aggregation and insolubilization, disrupt the associated functions, and ultimately contribute to the onset of senile cataract formation. General significanceThe mechanism underlying the onset of age-related diseases may involve isomerization, whereby d-amino acids are incorporated in the l-amino acid world of life.This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Toward proteome-scale identification and quantification of isoaspartyl residues in biological samples.

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins produce a mixture of aspartyl and isoaspartyl residues, the latter being involved in protein aging and inactivation. Electron capture dissociation (ECD) combined with Fourier transform mass spectrometry (FT MS) are known to be able to distinguish the isoaspartyl peptides by unique fragments of cn* + 58.0054 (C2H2O2) and z(l-n)-56.9976 (C2HO2), where n is the position of the aspartyl residue and l is the peptide length. In the present study, we tested the specificity of isoAsp detection using the accurate masses of the specific fragments. For this purpose, we analyzed 32 whole and partial proteomes obtained from human cells as well as tissue samples and identified by ECD 466 isoaspartyl peptide candidates. Detailed inspection revealed that many of these candidates were unreliable. To increase the isoAsp detection specificity, additional criteria had to be used, for example, adjacent c/z fragments, specific losses from the reduced species, and the shape of the chromatographic peak. Most stringent filtering of candidates yielded several cases where the presence of isoAsp was beyond doubt. Among the identified proteins with isoAsp, actin, heat shock cognate 71 kDa protein and pyruvate kinase have previously been identified as substrates for l-isoaspartyl methyltransferase, an important repair enzyme converting isoaspartyl to aspartyl. Quantification of relative isomerization degree was performed by the label-free approach. This is the first attempt to analyze the human isoaspartome in a high-throughput manner. The developed workflow allows for further enhancement of the detection rate of isoaspartyl residues in biological samples.

Deamidation: Differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins proceed through a succinimide intermediate producing a mixture of aspartyl and isoaspartyl residues. Isoaspartic acid is an isomer of aspartic acid with the C(beta) incorporated into the backbone, thus increasing the length of the protein backbone by one methylene unit. This post-translation modification is suspected to contribute to the aging of proteins and to protein folding disorders such as Alzheimer's disease, so that differentiating the two isomers becomes important. This manuscript reports that distinguishing aspartyl from isoaspartyl residues in peptides has been accomplished by electron capture dissociation (ECD) using a Fourier transform mass spectrometer (FTMS). Model peptides with aspartyl residues and their isoaspartyl analogs were examined and unique peaks corresponding to c(n)*+58 and z(l-n)-57 fragment ions (n, position of Asp; l, total number of amino acids in the peptide) were found only in the spectra of the peptides with isoaspartyl residues. The proposed fragmentation mechanism involves cleavage of the C(alpha)-C(beta) backbone bond, therefore splitting the isoaspartyl residue between the two fragments. Also, a complementary feature observed specific to aspartyl residues was the neutral loss of the aspartic acid side chain from the charge reduced species. CAD spectra of the peptides from the same instrument demonstrated the improved method because previously published CAD methods rely on the comparison to the spectra of standards with aspartyl residues. The potential use of the top-down approach to detect and resolve products from the deamidation of asparaginyl and isomerization of aspartyl residues is discussed.

Human Erythrocyte Proteinl-Isoaspartyl Methyltransferase: Heritability of Basal Activity and Genetic Polymorphism for Thermal Stability

Proteinl-isoaspartyl methyltransferase (PIMT) is believed to play an important role in the disposition of age-damaged proteins by catalyzing the repair of abnormal isoaspartyl linkages resulting from the spontaneous deamidation of asparaginyl residues or isomerization of aspartyl residues. As a step toward testing the hypothesis that human disease- or age-related pathology might be associated with a deficiency in PIMT, we investigated basal activity and thermal stability of PIMT in erythrocyte lysates from 299 U.S. family members. Thermal stability was measured because it is a sensitive measure of variation in amino acid sequence. Basal activity was normally distributed with a mean ± SD of 558 ± 43 units/ml erythrocytes. Statistical analysis of the data revealed that basal PIMT activity exhibited a high degree of heritability. Enzyme thermal stability showed a skewed bimodal frequency distribution, and segregation analysis of family member pedigrees was consistent with Mendelian inheritance of two major alleles. No DNA was available from the family samples, so we tested two additional population samples for a known Ile/Val polymorphism at codon 119 and for PIMT activity and thermal stability, using blood donated by 25 Norwegians and by 20 Koreans. Single-stranded conformational polymorphism analysis using polymerase chain reaction revealed a 100% correlation between thermal stability grouping and this polymorphism. The high thermal stability samples were all homozygous Ile, the low thermal stability samples were all homozygous Val, and the intermediate thermal stability samples were all heterozygous. Furthermore, this polymorphism was responsible, in part, for the variation observed in basal erythrocyte PIMT activity. These results will help provide a foundation for future studies aimed at correlating levels of PIMT activity, or other properties of this enzyme, with human disease.