Deuterium Labeling of Isoaspartic and Isoglutamic Acids for Mass Spectrometry Analysis.

Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid and has been linked to various diseases and health conditions. However, current methods for identifying isoAsp sites in proteins often suffer from ambiguity and have not gained widespread adoption. We developed a novel method that exclusively labels isoAsp with deuterium. This method capitalizes on the unique structural characteristics of isoAsp residues, which possess a free α-carboxyl group and can form an oxazolone ring. Once the oxazolone ring forms, it facilitates racemization at the Cα-position, incorporating a deuteron from a D2O solvent. The sites of deuterium-incorporated isoAsp in proteins can be unequivocally determined by comparing the precursor and product ion masses of the peptides from proteins reacted in H2O and D2O. The effectiveness of this method has been demonstrated through its application to model proteins lysozyme and rituximab. Furthermore, we have confirmed that the isoAsp deuterium-labeling reaction efficiently labels both l- and d-isoAsp without distinction, as well as isoglutamic acid (isoGlu), for which no effective detection methods currently exist.

Discrimination of Aspartic and Isoaspartic Acid Residues in Peptides by Tandem Mass Spectrometry with Hydrogen Attachment Dissociation.

Long-lived proteins undergo chemical modifications that can cause age-related diseases. Among these chemical modifications, isomerization is the most difficult to identify. Isomerization often occurs at the aspartic acid (Asp) residues. In this study, we used tandem mass spectrometry equipped with a newly developed ion activation method, hydrogen attachment dissociation (HAD), to analyze peptides containing Asp isomers. Although HAD preferentially produces [cn + 2H]+ and [zm + 2H]+ via N-Cα bond cleavage, [cn + 58 + 2H]+ and [zm - 58 + 2H]+ originate from the fragmentation of the isoAsp residue. Notably, [cn + 58 + 2H]+ and [zm - 58 + 2H]+ could be used as diagnostic fragment ions for the isoAsp residue because these fragment ions did not originate from the Asp residue. The detailed fragmentation mechanism was investigated by computational analysis using density functional theory. According to the results, hydrogen attachment to the carbonyl oxygen in the isoAsp residue results in the Cα-Cß bond cleavage. The experimental and theoretical joint study indicates that the present method allows us to discriminate Asp and isoAsp residues, including site identification of the isoAsp residue. Moreover, we demonstrated that the molar ratio of peptide isomers in the mixture could be estimated from their fragment ion abundance. Therefore, tandem mass spectrometry with HAD is a useful method for the rapid discrimination and semiquantitative analysis of peptides containing isoAsp residues.

Differentiation of Aspartic and Isoaspartic Acid Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid (isoAsp) is a ubiquitous modification that influences the structure and function of proteins. This modification of Asp impacts the stability of biotherapeutics and has been linked to the development of neurodegenerative diseases. We explored the use of 193 nm ultraviolet photodissociation (UVPD) to distinguish Asp and isoAsp in the protonated and deprotonated peptides. The differences in the relative abundances of several fragment ions uniquely generated by UVPD were used to differentiate isomeric peptide standards containing Asp or isoAsp. These fragment ions result from the cleavage of bonds N-terminal to Asp/isoAsp residues in addition to the side-chain losses from Asp/isoAsp or the losses of COOH, CO2, CO, or H2O from y-ions. Fragmentation of Asp-containing tryptic peptides using UVPD resulted in more enhanced w/w + 1/y - 1/x ions, while isoAsp-containing peptides yielded more enhanced y - 18/y - 45/y - 46 ions. UVPD was also used to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Moreover, UVPD of a protonated nontryptic peptide resulted in more enhanced y ions N- and C-terminal to isoAsp and differences in b/y ion ratios that were used to identify the isoAsp peptide.

Immunohistochemical Localization of D-ß-Aspartic Acid and Periostin in Vocal Fold Polyps.

Long-term voice abuse or sudden vocal fold microvascular disruption may lead to injury and subsequent repair/remodeling in the vocal fold mucosa. Periostin is known to be involved in airway remodeling and also in various otolaryngological diseases. D-ß-aspartic acid is the major isomer of D-aspartic acid found in elderly tissue. In this study we investigated the expression and the role of D-ß-aspartic acid and periostin in the formation of vocal fold polyps. The expression patterns of D-ß-aspartic acid and periostin in 36 surgical specimens of vocal fold polyps from 36 patients were investigated immunohistochemically. In the epithelium of vocal polyps, D-ß-aspartic acid was expressed in all cases. Expression of D-ß-aspartic acid was detected in 25 samples obtained from patients with vocal fold polyps stroma. Expression of periostin was detected in 28 samples obtained from patients with vocal fold polyps. Two patterns of D-ß-aspartic acid expression were observed in vocal fold polyps stroma: positive type and negative type. The following four patterns of periostin expression were observed in vocal fold polyps: negative type, superficial type, infiltrative type, and diffuse type. An association was observed between D-ß-aspartic acid expression patterns and periostin expression patterns. From these findings we speculate that periostin and D-ß-aspartic acid participate in certain pathological changes in vocal fold polyps, such as extracellular matrix accumulation, local fibrosis, and the formation and development of vocal fold polyps.

Testing the link between isoaspartate and Alzheimer's disease etiology.

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts protein structures, making them prone to aggregation. Here we strengthened the link between isoAsp and Alzheimer's disease (AD) by novel approaches to isoAsp analysis in human serum albumin (HSA), the most abundant blood protein and a major carrier of amyloid beta (Aß) and phosphorylated tau (p-tau) in blood. We discovered a reduced amount of anti-isoAsp antibodies (P < 0.0001), an elevated isoAsp level in HSA (P < 0.001), more HSA aggregates (P < 0.0001), and increased levels of free Aß (P < 0.01) in AD blood compared to controls. We also found that deamidation significantly reduces HSA capacity to bind with Aß and p-tau (P < 0.05). These suggest the presence in AD of a bottleneck in clearance of Aß and p-tau, leading to their increased concentrations in the brain and facilitating their aggregations there.

High-Resolution Demultiplexing (HRdm) Ion Mobility Spectrometry-Mass Spectrometry for Aspartic and Isoaspartic Acid Determination and Screening.

Isomeric peptide analyses are an analytical challenge of great importance to therapeutic monoclonal antibody and other biotherapeutic product development workflows. Aspartic acid (Asp, D) to isoaspartic acid (isoAsp, isoD) isomerization is a critical quality attribute (CQA) that requires careful control, monitoring, and quantitation during the drug discovery and production processes. While the formation of isoAsp has been implicated in a variety of disease states such as autoimmune diseases and several types of cancer, it is also understood that the formation of isoAsp results in a structural change impacting efficacy, potency, and immunogenic properties, all of which are undesirable. Currently, lengthy ultrahigh-performance liquid chromatography (UPLC) separations are coupled with MS for CQA analyses; however, these measurements often take over an hour and drastically limit analysis throughput. In this manuscript, drift tube ion mobility spectrometry-mass spectrometry (DTIMS-MS) and both a standard and high-resolution demultiplexing approach were utilized to study eight isomeric Asp and isoAsp peptide pairs. While the limited resolving power associated with the standard DTIMS analysis only separated three of the eight pairs, the application of HRdm distinguished seven of the eight and was only unable to separate DL and isoDL. The rapid high-throughput HRdm DTIMS-MS method was also interfaced with both flow injection and an automated solid phase extraction system to present the first application of HRdm for isoAsp and Asp assessment and demonstrate screening capabilities for isomeric peptides in complex samples, resulting in a workflow highly suitable for biopharmaceutical research needs.

Arabidopsis protein l-ISOASPARTYL METHYLTRANSFERASE repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance.

Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.

Protein-L-isoaspartate O-methyltransferase is required for in vivo control of oxidative damage in red blood cells.

Red blood cells have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which red blood cells neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how red blood cells repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to red blood cells is direct repair of the damaged molecules, as red blood cells cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. Red blood cells express an L-Isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters red blood cell metabolism in a healthy state, but does not impair the circulatory lifespan of red blood cells. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, red blood cells from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.

First Immunoassay for Measuring Isoaspartate in Human Serum Albumin.

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins mostly as a result of spontaneous deamidation of asparaginyl residues. An association has been found between isoAsp in human serum albumin (HSA) and Alzheimer's disease (AD). Here we report on a novel monoclonal antibody (mAb) 1A3 with excellent specificity to isoAsp in the functionally important domain of HSA. Based on 1A3 mAb, an indirect enzyme-linked immunosorbent assay (ELISA) was developed, and the isoAsp occupancy in 100 healthy plasma samples was quantified for the first time, providing the average value of (0.74 ± 0.13)%. These results suggest potential of isoAsp measurements for supplementary AD diagnostics as well as for assessing the freshness of stored donor blood and its suitability for transfusion.

An Unusually Rapid Protein Backbone Modification Stabilizes the Essential Bacterial Enzyme MurA.

Proteins are subject to spontaneous rearrangements of their backbones. Most prominently, asparagine and aspartate residues isomerize to their ß-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries. Such modifications are typically considered "molecular wear-and-tear", destroying protein function. However, the observation that some proteins, including the essential bacterial enzyme MurA, harbor stoichiometric amounts of isoAsp suggests that this modification can confer advantageous properties. Here, we demonstrate that nature exploits an isoAsp residue within a hairpin to stabilize MurA. We found that isoAsp formation in MurA is unusually rapid and critically dependent on folding status. Moreover, perturbation of the isoAsp-containing hairpin via site-directed mutagenesis causes aggregation of MurA variants. Structural mass spectrometry revealed that this effect is caused by local protein unfolding in MurA mutants. Our findings demonstrate that MurA evolved to "mature" via a spontaneous post-translational incorporation of a ß-amino acid, which raises the possibility that isoAsp-containing hairpins may serve as a structural motif of biological importance.

Functional divergence of annotated l-isoaspartate O-methyltransferases in an α-proteobacterium.

Spontaneous formation of isoaspartates (isoDs) often causes protein damage. l-Isoaspartate O-methyltransferase (PIMT) repairs isoD residues by catalyzing the formation of an unstable l-isoaspartyl methyl ester that spontaneously converts to an l-aspartyl residue. PIMTs are widely distributed in all three domains of life and have been studied most intensively in connection with their role in protein repair and aging in plants and animals. Studies of bacterial PIMTs have been limited to Escherichia coli, which has one PIMT. The α-proteobacterium Rhodopseudomonas palustris has three annotated PIMT genes, one of which (rpa2580) has been found to be important for cellular longevity in a growth-arrested state. However, the biochemical activities of these three R. palustris PIMTs are unknown. Here, we expressed and characterized all three annotated PIMT proteins, finding that two of them, RPA0376 and RPA2838, had PIMT activity, whereas RPA2580 did not. RPA0376 and RPA2838 single- and double-deletion mutants did not differ in longevity from WT R. palustris and did not exhibit elevated levels of isoD residues in aged cells. Comparative sequence analyses revealed that RPA2580 belongs to a separate phylogenetic group of annotated PIMT proteins present in the α-proteobacteria. Our results suggest that this group of proteins is not involved in repair of protein isoD residues. In addition, the bona fide bacterial PIMT enzymes may play a different or subtler role in bacterial physiology than previously suggested.

The l-isoaspartate modification within protein fragments in the aging lens can promote protein aggregation.

Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.

Negative ion cleavages of (M-H)- anions of peptides. Part 3. Post-translational modifications.

It is now 25 years since we commenced the study of the negative-ion fragmentations of peptides and we have recently concluded this research with investigations of the negative-ion chemistry of most post-translational functional groups. Our first negative-ion peptide review (Bowie, Brinkworth, & Dua, 2002) dealt with the characteristic backbone fragmentations and side-chain cleavages from (M-H)- ions of underivatized peptides, while the second (Bilusich & Bowie, 2009) included negative-ion backbone cleavages for Ser and Cys and some initial data on some post-translational groups including disulfides. This third and final review provides a brief summary of the major backbone and side chain cleavages outlined before (Bowie, Brinkworth, & Dua, 2002) and describes the quantum mechanical hydrogen tunneling associated with some proton transfers in enolate anion/enolate systems. The review then describes, in more depth, the negative-ion cleavages of the post-translational groups Kyn, isoAsp, pyroglu, disulfides, phosphates, and sulfates. Particular emphasis is devoted to disulfides (both intra- and intermolecular) and phosphates because of the extensive and spectacular anion chemistry shown by these groups. © 2016 Wiley Periodicals, Inc. Mass Spec Rev.

PIMT-Mediated Protein Repair: Mechanism and Implications.

Amino acids undergo many covalent modifications, but only few amino acid repair enzymes have been identified. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), also known as L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (PCMT), methylates covalently modified isoaspartate (isoAsp) residues accumulated in proteins via Asn deamidation and Asp hydrolysis. This cytoplasmic reaction occurs through the formation of succinimide cyclical intermediate and generates either isoAsp or Asp from succinimide. Succinimide conversion into Asp is spontaneous, while isoAsp is restored by PIMT using S-adenosylmethionine as a methyl donor. PIMT transforms isoAsp into succinimide, thereby creating an opportunity for the later to be converted into Asp. Apart from normal cell physiology, formation of isoAsp in proteins is promoted by various stress conditions. The resulting isoAsp can form a kink or bend in the protein backbone thus making the protein conformationally and functionally distorted. Many PIMT-interacting proteins (proteins with isoAsp residues) have been reported in eukaryotes, but only few of them have been found in prokaryotes. Extensive studies in mice have shown the importance of PIMT in neurodegeneration. Detail elucidation of PIMT function can create a platform for addressing various disorders such as Alzheimer's disease and cancer.

Polymorphic Variants of Human Protein l-Isoaspartyl Methyltransferase Affect Catalytic Activity, Aggregation, and Thermal Stability: IMPLICATIONS FOR THE ETIOLOGY OF NEUROLOGICAL DISORDERS AND COGNITIVE AGING.

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the human pcmt1 gene, catalyzes repair of abnormal l-isoaspartyl linkages in age-damaged proteins. Pcmt1 knock-out mice exhibit a profound neuropathology and die 30-60 days postnatal from an epileptic seizure. Here we express 15 reported variants of human PIMT and characterize them with regard to their enzymatic activity, thermal stability, and propensity to aggregation. One mutation, R36C, renders PIMT completely inactive, whereas two others, A7P and I58V, exhibit activity that is 80-100% higher than wild type. G175R is highly prone to aggregation and has greatly reduced activity. R17S and R17H show markedly enhanced sensitivity to thermal denaturation. Based on previous studies of moderate PIMT variation in humans and mice, we predict that heterozygosity for R36C, G175R, R17S, and R17H will prove detrimental to cognitive function and successful aging, whereas homozygosity (if it ever occurs) will lead to severe neurological problems in the young.

A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides.

Formation of isoaspartate (IsoAsp) from spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization is one of the most common non-enzymatic pathways of chemical degradation of protein and peptide pharmaceuticals. Rapid quantitation of IsoAsp formation can enable rank-ordering of potential drug candidates, mutants, and formulations as well as support shelf life prediction and stability requirements. A coupled enzymatic fluorescence-based IsoAsp assay (CEFIA) was developed as a high-throughput method for quantitation of IsoAsp in peptides and proteins. In this note, application of this method to two therapeutic candidate proteins with distinct structural scaffolds is described. In addition, the results obtained with this method are compared to those from conventional assays.

An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon IsoAsp formation but is restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE.

Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75's complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination.

Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.

Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH's (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.

Non-repair pathways for minimizing protein isoaspartyl damage in the yeast Saccharomyces cerevisiae.

The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50-300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.

Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and (3)H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼ 75-80 kDa, ∼ 95-100 kDa, and ∼ 155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼ 160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼ 2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.