Preparation of Affinity Purified Antibodies against ε-Glutaryl-Lysine Residues in Proteins for Investigation of Glutarylated Proteins in Animal Tissues

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.

Affinity sorbents for specific antibody purification were prepared by Affi-Gel 15 ("Bio-Rad Laboratories", Hercules, CA, USA) modification with ligands according to the producer manual. An amount of 13 mg of either ligand 1 or 2 (24 mmol each) were dissolved in 2 mL 0.1 M Na-Hepes buffer, 0.2 M NaCl, pH 7.5. Ice-cold ligand solutions were added each to a 1.6 mL portion of Affi-Gel 15 (preliminarily washed with the same buffer), mixed thoroughly and incubated 4 h at 4 • C. Ligand binding to the matrix was controlled via the filtrate reactivity with 1-fluor-2,4-dinitrobenzene after the reaction completion. Unreacted active matrix groups were blocked with tris(hydroxymethyl)aminomethane (Tris) upon slow washing of each portion of the matrix with 16 mL 0.1 M Tris-HCl buffer, pH 7.5. After that, the modified matrix was washed with 16 mL 20 mM phosphate buffer, pH 7.2 (PB), 10 mL PB with 1 M NaCl, 10 mL 0.15 M glycine-HCl buffer, pH 2.4, containing 10% 1,2-propanediol and equilibrated with PB. All buffers used for affinity chromatography contained 0.025% (w/v) NaN 3 .
Affinity purification of antibodies against glutarylated proteins. IgG fractions from immunized rabbit sera were prepared by ammonium sulfate fractionation followed by separation from other proteins on DEAE Blue agarose (DEAE Blue Affi-gel, «Bio-Rad Laboratories») [25,26] in 20 mM PB, pH 7.2.
Rabbit IgG fraction containing antibodies against glutaryl-GDH and glutaryl-BSA was applied to the affinity sorbent. An amount of 4 mL IgG fractions with protein concentration 300 µg/mL were slowly (for about 2 h) applied onto 1.6 mL columns with Affi-Gel 15 modified with either ligand 1 or ligand 2. After that, the columns were washed with 10 mL PB, 10 mL PB with 1 M NaCl. Ligand-bound antibodies were eluted with 8 mL 0.15 M glycine-HCl buffer, pH 2.4, containing 10% 1,2-propanediol. pH of eluted fractions was brought to 7.5-7.8 with 1 M Tris-HCl, pH 8.7 immediately after the elution.
Glutarylated proteins of the rat cerebral cortex or liver were determined via immunoblotting of homogenates of tissues of male rats and in extracts of mitochondrial fractions from these tissues prepared according to previously published protocols [27]. For immunoblotting, proteins were separated by SDS-PAGE according to Laemmli [21]. 2,2,2-Trichloroethanol was added to resolving gel containing 10% acrylamide for subsequent semi-quantitative determination of total protein by fluorescence under UV irradiation of the gel [28,29]. A total of 2 µg of the purified protein preparations or 22 µg of homogenate or mitochondrial extract total proteins was loaded onto the gel lanes. To study potential interconversion of the glutarylated bands resulting from endogenous proteolysis, incubation of the homogenates prior to the gel electrophoresis was performed at 37 • C for up to 3 h in a buffer, pH 8, containing 40 mM MOPS, 50 mM Tris-HCl, 0.15 mM EGTA, 4 mM MgCl 2 , 50 mM NaCl, 1 mM dithiothreitol, 20% glycerol, 0.75% Triton X-100 and protease inhibitors (0.15 mM AEBSF, 0.12 µM aprotinin, 2.5 µM bestatin, 2.25 µM E-64, 1.5 µM leupeptin and 1.05 µM pepstatin A).
After the electrophoresis, proteins were visualized in the gels by 2,2,2-trichloroethanolenhanced fluorescence and transferred onto a polyvinylidene fluoride (PVDF) membrane by semi-dry electrotransfer using a Power Blotter Station (Thermo Fisher Scientific) and Power Blotter 1-Step Transfer Buffer (Thermo Fisher Scientific) for 10 min at 2.5 A, 25 V. Immunovisualization of glutarylated proteins was performed using affinity purified antibodies as described above. The dilutions used were indicated in the figure captions. Anti-succinyl-lysine (PTM Biolabs #PTM-401, 1:2000) and anti-acetyl-lysine ("Cell Signaling Technologies" #9814, 1:1000) antibodies were used in a comparative analysis of antibodies against different types of lysine residue acylations.
Gels and membranes were analyzed using ChemiDoc MP System ("Bio-Rad Laboratories"). Mixtures of prestained (Bio-Rad #1610374, 1:7) and unstained strep-tagged (Bio-Rad #1610374, 1:120) marker proteins with known molecular weights were used to estimate the molecular weights of protein bands in the gel and on the membrane. Semi-quantitative estimation of luminescence (for glutarylated proteins) or fluorescence (for total protein) intensity of the stained bands was performed using the Image Lab software ("Bio-Rad Laboratories").
Protein concentrations in homogenates and mitochondrial extracts were determined by the Bradford method [30]; its micro variant [31] was used for the preparations of affinity purified antibodies.

Glutarylation of Proteins In Vitro
Commercial preparations of purified proteins (GDH from bovine liver, MDH from porcine heart and BSA) were modified with glutaric anhydride under the same conditions. The glutarylation under our conditions was confirmed by a comparative mass spectrometric analysis of peptide maps, using the samples of modified and unmodified GDH. A mass spectrometric analysis, carried out according to the published protocol [22], revealed a significant increase in the level of glutarylation of lysine residues in GDH upon treatment of the enzyme with glutaric anhydride. In the original GDH preparation, the degree of glutarylation of residues K171, K183, K187, K191, K390, K527 and K503 was very low compared to the modified protein. Treatment with glutaric anhydride led to a significant increase in the level of glutarylation of these and a number of other lysine residues in GDH. For example, the relative amount of the peptide-containing glutarylated lysine residue 503 (ISGASEKDIVHSGLAYTMER peptide; MS/MS spectra of glutarylated and non-glutarylated peptides are given in Supplement Figure S1) in the studied preparation of GDH, normalized to the sum of those for the well identifiable GDH peptides, not containing lysine residues (DSNYHLLMSVQESLER and DDGSWEVIEGYR) in the same preparation was increased by glutaric anhydride more than 10 times-from 0.002 ± 0.001 to 0.0320 ± 0.045 a.u. As noted earlier [22], these ratios were characterized by arbitrary units, which can be used for a comparative analysis of changes in the level of modification in the same enzyme preparation based on the determination of the same peptides. It should be kept in mind that such ratios do not characterize the percentage of the modified residue in the preparation, since the level of mass spectrometric identification of different peptides depends on many factors.
A comparison with published data [6] for a similar modification of GDH by a natural acylating agent, glutaryl-CoA, showed a noticeable, but incomplete, overlap of the enzyme lysine residues modified by both agents (Table 1). In overall, 9 and 12 peptides comprising the glutarylated lysine residues were determined after modification by 0.16 mM glutaric anhydride in this work and 0.5 mM glutaryl-CoA in an independent study [6]. Thus, the deeper modification was observed at the higher concentration of glutaryl-CoA compared to the lower concentration of glutaric anhydride. It is worth noting that the protein modification by glutaryl-CoA occurs through an intermediate formation of glutaric anhydride [32]. Hence, the observed differences in the modified peptides may manifest differences in the accessibilities and/or affinities of the residues in specific protein sites to the employed reagents. Moreover, the prolongated action of glutaric anhydride generated from glutaryl-CoA by intramolecular catalysis [32], compared to the less stable glutaric anhydride itself, may also contribute to the deeper modification in the former case compared to the latter one. Finally, an initial difference in the modification of specific residues in the two procedures may have caused different kinetics of GDH unfolding, exposing varied modifiable lysine residues during the reaction time. Table 1. Glutarylated lysine residues of the GDH peptides determined by mass spectrometry analysis of the purified enzyme after modification with 0.16 mM glutaric anhydride (this work) and 0.5 mM glutaryl-CoA [6]. * Lysine residue preceded by glycine residue.

Preparation and Characterization of Antibodies against Glutarylated Proteins
In our previous work, we used sequential immunization cycles with the replacement of the carrier protein at the same hapten to obtain anti-peptide antibodies that selectively interacted with one of the highly identical isoforms of the eukaryotic translation elongation factor 1A, 1A2 (eEF1A2) [26]. A similar approach was used to obtain antibodies to glutarylated proteins in the current work. For the first cycle of rabbit immunization, glutarylated GDH was employed and the second cycle of immunization was performed with the preparation of glutaryl-BSA. After the first cycle of immunization, a high antibody titer of 1:102,400 for glutaryl-GDH was determined, but the antibody titer for the non-glutarylated enzyme was just as high. The titer of antibodies against glutaryl-BSA in the rabbit antiserum obtained after the second cycle of immunization increased from 1:3200 (after the first cycle of immunization) to 1:102,400. At the same time, the titer of antibodies against glutaryl-GDH did not change and remained equal to 1:102,400, while the titer of antibodies against unmodified GDH decreased by 16 times. Thus, sequential cycles of immunization with two different glutarylated proteins increased the relative portion of the specific anti(ε-glutaryl-lysine) antibodies, not dependent of the carrier protein. Such a conclusion was confirmed below by results of the antibody affinity purification on the two different peptide ligands.
Two different affinity matrices with a lysine residue, glutarylated at its ε-amino-group, were prepared from Affi-Gel 15 for the affinity purification of the obtained antibodies. The first matrix comprised ligand one, ε-glutaryl-lysyl-glycinamide, and the second one comprised ligand two, glycyl-ε-glutaryl-lysinamide. Since lysine residues, which are subject to glutarylation, are usually located in the middle of peptide chains, the ligand amidation was used to mimic a peptide bond. The glycine residue played the same role; this residue was selected for its minimal size among amino acid residues. According to the results of the unbound ligand reactions with 1-fluoro-2,4-dinitrobenzene, the degree of conjugation of each ligand with the matrix was 14 micromoles per 1.6 mL of the matrix. Thus, the sorbents with ligands one and two differed in the location of the ε-glutaryl-lysine residue relative to the active carrier groups. Upon purification on columns of the same volume (1.6 mL), the ligand one-based column produced approximately twice as much IgG per chromatography cycle as the ligand-two-based column. Hence, the IgG yield from the ligand-one-based column was 26 µg IgG vs. An amount of 14 µg IgG from the ligand-one-based column. The fractions of antibodies not bound to these affinity matrices contained fairly large amounts of antibodies interacting with both glutarylated and nonglutarylated proteins (data not shown). The antibodies obtained by affinity purification on both the ligand-one-and ligand-two-incorporating matrices demonstrated selectivity for glutarylated proteins compared to the unmodified ones. Figure 1 compares the reactions of IgGs purified by affinity chromatography on matrices comprising ligand one or ligand two, from the antiserum obtained after the two immunization cycles, with glutarylated and non-glutarylated GDH, MDH and BSA. No significant differences in the selectivity of both types of antibodies against glutarylated proteins in the range of optimal concentrations for immunoblotting (0.01-0.13 µg/mL) were obvious. In further experiments, we used antibodies purified on the ligand-one-comprising matrix, which was characterized by a higher IgG yield.
Thus, highly selective antibodies towards glutarylated lysine residues were obtained after the two immunization cycles (Figure 1). Some reactivity observed at the high concentrations of antibodies against unmodified commercial GDH and BSA preparations may have resulted from a low level of the known physiological glutarylation of the enzyme and a large amount of the purified protein used for this assay. As indicated above, we identified a low level of glutarylation of a number of lysine residues in the non-treated preparation of purified liver GDH, which was also observed by others [6]. Some glutarylation of the commercial BSA preparations was likely in view of the known glutarylation of human serum albumin [9], although this modification was not detectable in BSA in an independent study [32]. So, the antibody interaction with unmodified BSA may have been caused by either its very low glutarylation level, or unspecific interaction. Nevertheless, Figure 1 demonstrates a minor contribution of this immunostaining, compared to that of the glutarylated BSA, especially under the optimal concentration of the antibodies and the affinity purification employed on ligand one. MDH glutarylation in the glutaric-anhydride-treated samples was the least pronounced. Thus, the choice of ε-glutaryl-lysine residues within the peptide backbone as affinity ligands was successful for the purification of antibodies that specifically interacted with glutarylated proteins. Thus, highly selective antibodies towards glutarylated lysine residues were obtained after the two immunization cycles (Figure 1). Some reactivity observed at the high concentrations of antibodies against unmodified commercial GDH and BSA preparations may have resulted from a low level of the known physiological glutarylation of the enzyme and a large amount of the purified protein used for this assay. As indicated above, we identified a low level of glutarylation of a number of lysine residues in the non-treated preparation of purified liver GDH, which was also observed by others [6]. Some glutarylation of the commercial BSA preparations was likely in view of the known glutarylation of human serum albumin [9], although this modification was not detectable in BSA in an independent study [32]. So, the antibody interaction with unmodified BSA may have been caused by either its very low glutarylation level, or unspecific interaction. Nevertheless, Figure 1 demonstrates a minor contribution of this immunostaining, compared to that of the glutarylated BSA, especially under the optimal concentration of the antibodies and the affinity purification employed on ligand one. MDH glutarylation in the glutaric-anhydride-treated samples was the least pronounced. Thus, the choice of ε-glutaryl-lysine residues within the peptide backbone as affinity ligands was successful for the purification of antibodies that specifically interacted with glutarylated proteins.
It should be noted that glycine residues was adjacent to 4 out of 32 lysine residues in bovine GDH, 4 out of 60 lysine residues in BSA, 2 out of 31 lysine residues in porcine cytoplasmic MDH and 9 out of 25 lysine residues in porcine mitochondrial MDH. Taking into account the lowest reactivity of glutarylated MDH out of the three studied proteins to the antibodies (Figure 1) and the fact that only one of the nine mass-spectrometry-identified ε-glutarylated GDH lysine residues was adjacent to the glycine residue (K480 , Table 1), the interaction of the obtained antibodies with glutarylated proteins was determined solely by the ε-glutarylation of their lysine residues, and It should be noted that glycine residues was adjacent to 4 out of 32 lysine residues in bovine GDH, 4 out of 60 lysine residues in BSA, 2 out of 31 lysine residues in porcine cytoplasmic MDH and 9 out of 25 lysine residues in porcine mitochondrial MDH. Taking into account the lowest reactivity of glutarylated MDH out of the three studied proteins to the antibodies (Figure 1) and the fact that only one of the nine mass-spectrometry-identified ε-glutarylated GDH lysine residues was adjacent to the glycine residue (K480, Table 1), the interaction of the obtained antibodies with glutarylated proteins was determined solely by the ε-glutarylation of their lysine residues, and not by the proximity of ε-glutaryl-lysine to glycine residues realized in the affinity ligand.
It is worth noting that increasing concentrations of interacting partners, such as the glutarylated proteins and anti(ε-glutaryl-lysine) antibodies, inevitably increases non-specific interactions, which may contribute to the observed minor interaction of the affinity-purified anti(ε-glutaryl-lysine) antibodies with unmodified BSA and MDH at a high concentration of the antibodies, in addition to the potential endogenous glutarylation (Figure 1, lower panels at the same intensity for all dots). In fact, at the optimal dilution, both the homemade and commercial anti(ε-glutaryl-lysine) antibodies, showed a minor reactivity to the non-glutarylated GDH, with such a reactivity of BSA being negligible. This corresponded to the known glutarylation of GDH which was detected also in the current study by our MS procedure, and there was no MS-detectable glutarylation of the BSA preparation [32]. Remarkably, both with the home-made and commercial antibodies, the reactivity of the nonglutarylated GDH to anti(ε-glutaryl-lysine) antibodies did not change after the acetylation or succinylation of GDH, but strongly increased after the enzyme glutarylation ( Figure 2). Additionally, with BSA, only glutarylation, but not acetylation or succinylation, strongly increased its reactivity to both our and commercial anti(ε-glutaryl-lysine antibodies. This indicates the antibodies selectivity towards glutarylated lysine residues in proteins. the BSA preparation [32]. Remarkably, both with the home-made and commercial antibodies, the reactivity of the non-glutarylated GDH to anti(ε-glutaryl-lysine) antibodies did not change after the acetylation or succinylation of GDH, but strongly increased after the enzyme glutarylation ( Figure 2). Additionally, with BSA, only glutarylation, but not acetylation or succinylation, strongly increased its reactivity to both our and commercial anti(ε-glutaryl-lysine antibodies. This indicates the antibodies selectivity towards glutarylated lysine residues in proteins.

Figure 2.
Comparison of specificity and selectivity of the home-made and commercial anti-(ε-glutaryl-lysine) antibodies, using purified proteins with artificially modified lysine residues (see details in Materials and Methods). An amount of 1.5 µg/mL concentration of commercial antibodies represents the one recommended for immunoblotting by the manufacturer.
Although the specificity of the obtained antibodies towards ε-glutaryl-lysine residues of different proteins allows one to use them for the determination of glutarylated proteins in animal tissues, one should keep in mind that the degree of the protein glutarylation under physiological conditions is not comparable to that after the chemical modification by glutarylation agents [6]. Even for the most common acyl modification of lysine residues, i.e., acetylation, the extent of modification is far from 100% and depends on the physiological state and specific features of tissue metabolism [17,22]. Therefore, the antibody concentrations for the determination of purified proteins, artificially glu- Although the specificity of the obtained antibodies towards ε-glutaryl-lysine residues of different proteins allows one to use them for the determination of glutarylated proteins in animal tissues, one should keep in mind that the degree of the protein glutarylation under physiological conditions is not comparable to that after the chemical modification by glutarylation agents [6]. Even for the most common acyl modification of lysine residues, i.e., acetylation, the extent of modification is far from 100% and depends on the physiological state and specific features of tissue metabolism [17,22]. Therefore, the antibody concentrations for the determination of purified proteins, artificially glutarylated with a high degree of modification, were not applicable for the analysis of protein glutarylation in the complex biological samples. In fact, Figure 3 shows that, despite the intense staining of glutarylated GDH and BSA, the used concentration of the anti-(ε-glutaryl-lysine) antibodies (0.016 µg/mL) hardly detected glutarylated proteins in extracts of liver mitochondria.
In this case, the determination of glutarylated proteins in the extract required a longer exposure and/or image contrasting (Figure 3a; lanes 8a and 17a which are presented separately). Nevertheless, this concentration of antibodies was enough to visualize the mitochondrial proteins after their artificial modification with glutaric anhydride (Figure 3a; gel lanes 9 and 18). At the same time, a comparison of the native and glutarylated extracts of the liver mitochondrial fraction showed that treatment with glutaric anhydride not only led to the appearance of multiple bands that were absent in the non-treated extract, but also increased the glutarylation of proteins that were relatively weakly glutarylated under native conditions (protein bands with molecular weights of 75 and 130 kDa, Figure 3). This experiment demonstrated both a low level of modification of potentially glutarylated proteins under native conditions and a rather high specificity of the obtained anti-(εglutaryl-lysine) antibodies towards this modification, regardless of the particular protein.
tarylated with a high degree of modification, were not applicable for the analysis of protein glutarylation in the complex biological samples. In fact, Figure 3 shows that, despite the intense staining of glutarylated GDH and BSA, the used concentration of the anti-(ε-glutaryl-lysine) antibodies (0.016 µg/mL) hardly detected glutarylated proteins in extracts of liver mitochondria.

Characterization of Protein Glutarylation in the Rat Brain and Liver Homogenates
The obtained anti-(ε-glutaryl-lysine) antibodies were used to detect glutarylated proteins in the rat brain and liver homogenates. As can be seen from Figure 4, brain homogenates exhibited one major glutarylated protein band with a characteristic molecular weight of 75 kDa, and several minor ones. The pattern of protein glutarylation in the liver homogenates differed from that in the brain homogenates (Figure 4), with more glutarylated bands observed in the former than in the latter sample. Two of the main fractions of glutarylated proteins in the liver homogenates were characterized by the same molecular weights (75 and 130 kDa) as the glutarylated proteins determined in the mitochondrial extract (Figure 2). It should be noted that none of the identified fractions coincided in molecular weight with glutaryl-GDH (55 kDa) and glutaryl-BSA (69 kDa), the bands of which were present on the immunoblot of purified proteins (Figure 3) in the corresponding region between the 50 kDa and 75 kDa markers. The failure to detect glutarylated GDH in brain extracts from control rats was consistent with the absence of noticeable GDH glutarylation in the murine brain under physiological conditions [6]. Our mass spectrometric identification of rat GDH and serum albumin in brain homogenates also did not reveal their glutarylated fragments, although serum albumin was abundant in these samples: up to 60 peptides covering 65% of the serum albumin sequence were determined in the rat brain homogenates. This was consistent with a good separation of the protein bands corresponding to 69 kDa serum albumin and 75 kDa glutarylated protein (Figures 3 and 4), providing additional evidence that antibody staining of the 75 kDa band in the rat brain and liver homogenates did not originate from the reaction with albumin.
Incubation of homogenates at 37 • C resulted in a redistribution of the intensity of several glutarylated protein bands. The observed changes indicated the possibility of endogenous proteolysis, despite the presence of protease inhibitors in the homogenates. Thus, after a 3 h incubation of the brain homogenates, the intensity of the low-molecularweight band at the 20 kDa level increased, simultaneously with a decrease in the highmolecular-weight region (at 90 kDa level) of the 75 kDa protein peak (Figure 3c). Similarly, in the liver homogenate, the intensity of the protein band with a molecular weight of about 130 kDa decreased with a simultaneous increase in the intensity of the 75 kDa band. Thus, the possibility of endogenous proteolysis should be taken into account during the long-term incubations of the homogenates, which is often used in the studies of various factors potentially regulating protein glutarylation. Accompanying proteolytic degradation may result in an apparent increase or decrease in the intensities of the glutarylated protein bands due to the changes in the amount of protein in these bands.

Specific Patterns of Glutarylation, Succinylation and Acetylation in Biological Preparations
The degree of possible cross-interaction of the obtained antibodies with lysine residues modified by other acyl species was assessed by comparing the immunoblotting patterns of the same biological samples (rat brain homogenates) obtained using antibodies against lysine glutarylation, succinylation and acetylation ( Figure 4).
As seen from Figure 5, the visualization of glutaryl-, succinyl-and acetyl-lysine residues of rat cerebral cortex proteins with appropriate antibodies revealed completely different patterns of the distribution of such residues over various proteins. For example, the main band of glutarylated proteins was characterized by a molecular weight of 75 kDa. The strongest staining of succinyllysine residues was observed in proteins having molecular weights of 32, 38 and 40 kDa. The main band of acetyl-lysine-containing proteins was 51 kDa. It should be noted that proteins of similar molecular weights as well as the same protein may undergo different types of acylation. Therefore, a staining of individual protein bands with different types of antibodies may be observed. For example, proteins in the region below 20 kDa, corresponding to histones undergoing various modifications affecting their electrophoretic mobility [3,11,12,15], may react with various anti-acyl-lysine antibodies. Nevertheless, the different overall patterns of specific acylation types, presented in Figure 5, confirmed the high specificity of the used antibodies towards the corresponding types of post-translational acylation of protein lysine residues.

Conclusions
The anti-(ε-glutaryl-lysine) antibodies obtained in this work after the two cycles of immunization and subsequent affinity purification on specific ligands enabled the specific detection of glutarylated proteins by immunoblotting of animal tissue preparations. These antibodies were reactive towards various glutarylated proteins. In addition to glutaryl-GDH and glutaryl-BSA used for immunization, the antibodies also interacted with the purified preparation of glutarylated MDH, as well as with glutarylated proteins of rat brain and liver homogenates, whose molecular weights were different from those of GDH and BSA. Cross-reactions with the non-modified proteins were mostly limited to endogenously glutarylated proteins and were not observed with the proteins after in vitro succinylation or acetylation. This selectivity provides a difference in the patterns of protein glutarylation, succinylation and acetylation in rat brain cortex homogenates, revealed by the antibodies of corresponding specificities. The comparative analysis of these patterns demonstrates the efficiency of the elaborated antibodies to detect specific types of protein acylations in complex biological samples. Immunoblotting of proteins of the rat brain and liver homogenates or mitochondria using the obtained anti-(ε-glutaryl-lysine) antibodies revealed a low level of proteins glutarylation in these tissues and subcellular fractions. In tissue homogenates, high-molecular-weight glutarylated proteins may undergo proteolytic fragmentation which must be taken into account when assessing and comparing glutarylation patterns.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure  S1: Representative MS/MS spectra of the GDH peptides containing K503 residue.
Author Contributions: Conceptualization, V.I.B.; methodology, all co-authors; validation, all co-authors; formal analysis, all co-authors; investigation, A.V.A., E.F.K., A.I.B., S.N.G., A.A.C., Figure 5. Visualization of glutarylation, succinylation and acetylation of proteins of the brain cortex of the male rats aged 10-12 weeks. Proteins were separated by SDS-PAGE and visualized on the membrane using the obtained antibodies against ε-glutaryl-lysine (0.8 µg/mL) and commercial antibodies against ε-succinyl-lysine (0.5 µg/mL) and ε-acetyl-lysine (0.29 µg/mL). (A) Immunoblotting of the cerebral cortex samples from male rats; 1-4-different animals; M-protein markers with known molecular weights indicated to the left of the blots. (B) Densitograms of the results of immunoblotting with automatically determined molecular weights of the major protein bands.

Conclusions
The anti-(ε-glutaryl-lysine) antibodies obtained in this work after the two cycles of immunization and subsequent affinity purification on specific ligands enabled the specific detection of glutarylated proteins by immunoblotting of animal tissue preparations. These antibodies were reactive towards various glutarylated proteins. In addition to glutaryl-GDH and glutaryl-BSA used for immunization, the antibodies also interacted with the purified preparation of glutarylated MDH, as well as with glutarylated proteins of rat brain and liver homogenates, whose molecular weights were different from those of GDH and BSA. Cross-reactions with the non-modified proteins were mostly limited to endogenously glutarylated proteins and were not observed with the proteins after in vitro succinylation or acetylation. This selectivity provides a difference in the patterns of protein glutarylation, succinylation and acetylation in rat brain cortex homogenates, revealed by the antibodies of corresponding specificities. The comparative analysis of these patterns demonstrates the efficiency of the elaborated antibodies to detect specific types of protein acylations in complex biological samples. Immunoblotting of proteins of the rat brain and liver homogenates or mitochondria using the obtained anti-(ε-glutaryl-lysine) antibodies revealed a low level of proteins glutarylation in these tissues and subcellular fractions. In tissue homogenates, high-molecular-weight glutarylated proteins may undergo proteolytic fragmentation which must be taken into account when assessing and comparing glutarylation patterns.