Efficient Enrichment Method for N-Phosphorylation Peptides in Mouse Brain Tissue

Abstract

In addition to O-phosphorylation, protein N-phosphorylation was proven to play important roles in multiple biological processes. Although affinity methods were developed for the enrichment of N-phosphorylation peptides in cells, it was still difficult to enrich N-phosphorylation peptides in tissue due to its complexity. In this study, we established a workflow for the identification of N-phosphorylation peptides in mouse brain tissue by direct enrichment in high concentration urea. In total, 989 N-phosphorylation sites were obtained using 0.5 M urea as enrichment buffer. Among all identified N-phosphorylation sites, the localization probability over 0.75 was as high as 80%, suggesting the reliability of the method. Furthermore, the sequence motif analysis and gene ontology analysis results showed a good match to previous studies. The method was successfully used for N-phosphorylation analysis of mouse hippocampus from Alzheimer’s disease model, and 533 N-phosphorylation sites were identified in 5 × FAD mouse hippocampus tissue. Biological process analysis results showed that “brain development”, “cellular response to reactive oxygen species”, “microtubule cytoskeleton organization”, and “peptidyl-serine phosphorylation” were especially enriched in 5 × FAD mouse. It is suggested that N-phosphorylation may be related to Alzheimer’s disease in these aspects.

Mass Spectrometry in China

“…Due to the high chemical diversity and complexity, post-translational modification (PTM) characterization in proteomics still has many unsolved problems. Zhao, Jiang et al developed a method for fast enrichment of N-phosphorylation peptides in mouse brain tissue using high concentration urea, which led to the identification of 989 N-phosphorylation sites . Sun et al used newly developed software, StrucGP, to profile the structure and N-glycosylation on the COVID-19 virus spike protein …”

“…Zhao, Jiang et al developed a method for fast enrichment of N-phosphorylation peptides in mouse brain tissue using high concentration urea, which led to the identification of 989 N-phosphorylation sites. 40 Sun et al used newly developed software, StrucGP, to profile the structure and N-glycosylation on the COVID-19 virus spike protein. 41 Structural proteomics is an emerging area of proteomics research investigating protein higher-order structures and noncovalent interactions.…”

Anti‐solvent method synthesized phosphorylated microcapsules with flame retardancy and self‐healing analysis

“…Functionalization of the cellulose shell by phosphoric acid groups will confer excellent flame-retardant properties, thermal stability, and biocompatibility to cellulose. [22][23][24][25] Kokol et al 26 used phosphoric acid as the phosphoryl donor and employed molten urea as the reaction medium under homogeneous conditions to synthesize cellulose esters with phosphoryl side groups, namely, p-NFC and p-NCC, from nanofibrillated cellulose (NFC) and nanocrystalline cellulose (NCC). Zhang et al 27 used the ionic liquid 1,3-dimethylimidazolium trimethylphosphate as a modifier to prepare flame-retardant phosphorylated lyocell fibers (FRLF).The results of microscale combustion calorimetry showed that, compared to single fibers (LF), the peak heat release rate (PHRR) of FRLF decreased by 50.9%, demonstrating excellent flame retardancy.…”

“…This dual functionality is crucial for enhancing the material's durability, safety, and operational efficiency. Functionalization of the cellulose shell by phosphoric acid groups will confer excellent flame‐retardant properties, thermal stability, and biocompatibility to cellulose 22–25 . Kokol et al 26 used phosphoric acid as the phosphoryl donor and employed molten urea as the reaction medium under homogeneous conditions to synthesize cellulose esters with phosphoryl side groups, namely, p‐NFC and p‐NCC, from nanofibrillated cellulose (NFC) and nanocrystalline cellulose (NCC).…”