**3. DPM studies in brain tissue from dementia patients**

DPMs caused by spontaneous chemical reactions can radically alter protein structure and function, promoting pathological progression. Key DPMs including oxidation, deamidation, racemization, glycation, advanced glycation end products, and enzymatic modifications such as citrullination typically alter the charged state and hydrophobicity of the affected protein. This changes charge and hydrophobic nature of protein-promoting protein misfolding and aggregation. Despite the clinical importance of DPMs in neurodegenerative diseases, the mechanism and cause of modifications are poorly understood, largely due to the technical challenges. To define the role of DMPs, accurate identification of protein modification sites is important. However, it is important to avoid the introduction of artificial modification during sample preparation and improve sensitivity and confidence of identifying low-abundant modifications. According to Hao et al. [27, 31], processing proteomic samples at a mild alkaline pH and prolonged incubation at 37°C during trypsin digestion were major causes of nonen‐ zymatic asparagines (Asn)-deamidation. Therefore, these researchers proposed an improved protocol of trypsin digestion in 50 mM ammonium acetate (pH 6) to avoid introduction of artifactual deamidation during sample preparation. Moreover, a sodium deoxycholate (SDC) and ammonium acetate-based buffer (pH 6.5) have been developed to increase protein solubility, to enhance trypsin activity, and to improve the recovery of low-abundant peptides from complex biological samples. This mildly acidic conditions and absence of urea minimized artificial asparagine deamidation and prevented artifactual carbamylation [61].

Under physiological conditions, deamidation of the protein residues asparagines (Asn) and glutamine (Gln) can occur spontaneously and progressively alters protein structure, function, and stability over time. Asn deamidation occurs through the formation of a succinimide ring intermediate, which quickly gets hydrolyzed to D,L-Asp and D,L-isoAsp with isoAsp predominating. Deamidation of Gln occurs much slower since it is thermodynamically less favorable to form a six-membered glutarimide ring. Deamidation causes an increase in the mass of 0.984 Da. The separation of Asp- and isoAsp remains challenging since peptides containing Asp- and isoAsp display the similar mass and hydrophobicity. However, improved ERLIC-LC-MS/MS method allows distinguishing isoAsp-containing peptide from n-Aspcontaining peptide prior to their identification. Protein deamidation serves as a versatile molecular clock that can regulates many biological processes. Protein modifications and their biological impacts have been recently reviewed by Hao et al. [27]. Proteins with low turnover rates accumulate nonenzymatic modifications that cannot be repaired, and thus these modi‐ fications including deamidation cause age-related changes in biological functions and play major role in aging. Deamidation has been linked with alterations in the structure of human cortical neurons [62]. An accumulation of protein α-synuclein is a pathological characteristic of dementia with Lewy bodies (DLB), PD, AD, and multiple system atrophy (MSA), and can be linked to protein deamidation. The excessive deposition of IsoAsp residues in synapsin 1 and tubulin proteins in VaD [30] suggests that deamidation of synaptic proteins impairs its function and may cause dementia.

#### **3.1. Deamidation of ion channel and other proteins in dementia**

investigators [53–57] have yielded a good progress in understanding how protein DPMs, and protein aggregation induced by hypoxic-ischemic brain injury can promote neurodegenera‐ tion in dementia. This "vicious cycle" of brain tissue damage is summarized in **Figure 2**.

116 Update on Dementia

**Figure 2.** Vicious cycle of hypoxia-ischemic brain injury, degenerative protein modifications, and amyloidosis.

DPMs caused by spontaneous chemical reactions can radically alter protein structure and function, promoting pathological progression. Key DPMs including oxidation, deamidation, racemization, glycation, advanced glycation end products, and enzymatic modifications such as citrullination typically alter the charged state and hydrophobicity of the affected protein. This changes charge and hydrophobic nature of protein-promoting protein misfolding and aggregation. Despite the clinical importance of DPMs in neurodegenerative diseases, the mechanism and cause of modifications are poorly understood, largely due to the technical challenges. To define the role of DMPs, accurate identification of protein modification sites is important. However, it is important to avoid the introduction of artificial modification during sample preparation and improve sensitivity and confidence of identifying low-abundant modifications. According to Hao et al. [27, 31], processing proteomic samples at a mild alkaline pH and prolonged incubation at 37°C during trypsin digestion were major causes of nonen‐ zymatic asparagines (Asn)-deamidation. Therefore, these researchers proposed an improved protocol of trypsin digestion in 50 mM ammonium acetate (pH 6) to avoid introduction of artifactual deamidation during sample preparation. Moreover, a sodium deoxycholate (SDC) and ammonium acetate-based buffer (pH 6.5) have been developed to increase protein solubility, to enhance trypsin activity, and to improve the recovery of low-abundant peptides from complex biological samples. This mildly acidic conditions and absence of urea minimized

**3. DPM studies in brain tissue from dementia patients**

artificial asparagine deamidation and prevented artifactual carbamylation [61].

The ion channel protein Na+ −K<sup>+</sup> −ATPase exhibits multiple functions including the mainte‐ nance of differential membrane potential in neurons, which is an essential feature of the signal transduction processes. Dysregulation of Na+ −K<sup>+</sup> −ATPase expression or function has been reported in both animal model and human brain tissues affected by AD, PD, and HD. In the study of human brain tissues from patients with VaD, Adav et al. [24] noted deamidation of Na+ −K<sup>+</sup> −ATPase subunits in the evolutionary-conserved regions. Using structural model, they located the modification sites and proposed that the disruption of Mg2+− and Cu2+−binding sites impaired electrostatic interactions and function of ion channel proteins in VaD (**Fig‐ ure 3**). Modification of residues 210 and 220 has been proposed to cause defects in protein phosphorylation and dephosphorylation mechanisms, leading to altered ATP hydrolysis. Deamidation-induced changes in Na+ −K<sup>+</sup> −ATPase subunit proteins may lead to defects in membrane excitability and neuronal function. Moreover, the enzyme "protein L-isoaspartate (D-aspartate) O-methyltransferase" (PIMT) functions as a protein repair enzyme and has the potential to recognize these abnormal residues (isoAsp) and convert them to the normal L-Asp form. Thus, deamidation can be repaired. However, according to proteomic analysis of VaD brain tissues, PIMT was also deamidated. Deamidation of PIMT could manipulate its potential to recognize abnormal residues or impair its potential to convert isoaspartyl to the normal Laspartyl form [24]. In mammalian cells and mouse models lacking repair enzyme PIMT, isoASP accumulation causes hyperactivation of key cell-signaling pathways, weakening animal growth and even fatal seizures [63].

During the characterization of the human brain amyloidal plaque from dementia patients, deamidation of aggregated proteins was noted. The extensively deamidated proteins were S100A9, ferritine, and hemoglobin. In addition to these proteins, proteins such as S100 calciumbinding protein B (S100-B), α2(IV), and α2(I) chains of human collagen, extracellular matrix such as laminin subunit β-2 was found to be deamidated. Further, these authors found deamidated adhesion junction plaque protein dystonin (isoform 3) and many others [34]. Proteins coronin-1A and syntaxin-binding protein 2, which were previously been implicated in the neurodegeneration of the hippocampus, were also found deamidated detected in brain tissue sample of demented patients. Deamidation introduces negative charge at sites of modification. This change in charge promotes protein aggregation and remains as a patho‐ logical hallmark of age-related disorders and neurodegenerative diseases. Thus, the multiple deamidated residues of S100A9 (**Figure 3C** and **D**) could introduce a negative charge to form pathological aggregates in the brain. Hence, an accurate identification of DMPs and modifi‐ cation sites is important to understand the role of DPMs in human diseases. A comprehensive investigation including method development for accurate identification of DPMs has been performed for biomedical research [24, 26, 27, 30, 31, 34, 35].

**Figure 3.** Structural models of Na+ /K+ -ATPase catalytic site in (A) E1P (PDB ID 4HQJ) and (B) E2P (2ZXE). Domain A is shown in blue and cyan color, domain P is shown in yellow, while domain N is shown in green. The deamidation sites (N210, D220, and N715) are shown in red color (adapted with permission from Adav et al. [24]). The deamidation sites of protein S100A9 (RCSB Protein Data Bank accession code: 1XK4) are shown in (C). EF hands have been displayed in yellow color and deamidation sites in magenta and blue. EF hands alone are shown in (D).

Loss of synapses is one of the most significant contributors to the cognitive impairment manifest in VaD and other neurodegenerative diseases. Following synapses loss, the remain‐ ing synapses alter their shape. According to recent literature [26], synaptic immunoglobulins were perturbed proteins in VaD temporal cortices, while SNAP25 was substantially upregulated. Further, deamidation studies revealed that the protein synapsin 1 displayed significant accumulation of deamidated asparagine and glutamine residues when compared with age-matched control [30]. The location of the modification site using structural model demonstrated that the deamidation sites in synapsin 1 were likely to induce pathological changes in protein conformation.
