Brain Proteomics Supports the Role of Glutamate Metabolism and Suggests Other Metabolic Alterations in Protein l-Isoaspartyl Methyltransferase (PIMT)-Knockout Mice

Abstract

Protein L-isoaspartyl methyltransferase (PIMT) repairs the isoaspartyl residues (isoAsp) that originate from asparagine deamidation and aspartic acid (Asp) isomerization to Asp residues. Deletion of the gene encoding PIMT in mice (Pcmt1) leads to isoAsp accumulation in all tissues measured, especially in the brain. These PIMT-knockout (PIMT-KO) mice have perturbed glutamate metabolism and die prematurely of epileptic seizures. To further elucidate the role of PIMT, brain proteomes of PIMT-KO mice and controls were analyzed. The isoAsp levels from two of the detected 67 isoAsp sites (residue 98 from calmodulin and 68 from glyceraldehyde-3-phosphate dehydrogenase) were quantified, and found significantly increased in PIMT-KO mice (p < 0.01). Additionally, the abundance of at least 151 out of the 1017 quantified proteins was found to be altered in PIMT-KO mouse brain. Gene Ontology analysis revealed that many down-regulated proteins are involved in cellular amino acid biosynthesis. For example, the serine synthesis pathway was suppressed, possibly leading to reduced serine production in PIMT-KO mice. Additionally, the abundances of enzymes in the glutamate-glutamine cycle were altered towards the accumulation of glutamate. These findings support the involvement of PIMT in glutamate metabolism and suggest that the absence of PIMT also affects other processes involving amino acids synthesis and metabolism.

Development of an Automated, High‐throughput Sample Preparation Protocol for Proteomics Analysis

“…Proteolytic products pass through the membrane filter because their size is usually <5 kDa. Because of its versatility, FASP has been applied in numerous proteomics studies . However, application of FASP for high‐throughput data analysis is very limited and difficult because of the involvement of many washing and transfer steps, with centrifugation being the major disadvantage.…”

“…Because of its versatility, FASP has been applied in numerous proteomics studies. [21][22][23][24] However, application of FASP for high-throughput data analysis is very limited and difficult because of the involvement of many washing and transfer steps, with centrifugation being the major disadvantage. Because centrifugation takes longer time and needs the transfer the filter plate from one place to another, it may result in sample loss or degradation.…”

Strategies for analysis of isomeric peptides

“…While isoAsp residues could both be found in the wild‐type and knock‐out model, 23 additional sites of isoAsp formation were found solely in the knock‐out model. Reprinted with permission from the American Chemical Society …”

“…While isoAsp residues could both be found in the wild-type and knock-out model, 23 additional sites of isoAsp formation were found solely in the knock-out model. Reprinted with permission from the American Chemical Society [62] copolymerization of lauryl methacrylate, and ethylene dimethacylate with CE, that was used to separate several isomeric peptides. The developed method provided a separation that was previously had not been possible with CE [49].…”

Recent advances in mass spectrometric analysis of protein deamidation

“…Tissue accumulation of IsoAsp‐containing proteins in PIMT knockout (KO) mice is time‐dependent, and therefore mimics the natural proteomics of aging and associated pathologies (Clarke, ; Reissner & Aswad, ). When compared with wild‐type mice, the brain proteome of PIMT‐KO animals was found to include 67 novel isoAsp sites and increased levels of isoAsp motifs at residue 98 in calmodulin and residue 68 in the enzyme glyceraldehyde‐3‐phosphate dehydrogenase (Yang et al, ). The brain proteomes of PIMT‐KO mice also exhibit sex‐specific differences, including greater phosphorylation of synapsins I and II in females (Qin et al, ), which resembles the gender bias of mixed dementia in human women.…”