Recent advances in mass spectrometric analysis of protein deamidation

Abstract

Protein deamidation has been proposed to represent a "molecular clock" that progressively disrupts protein structure and function in human degenerative diseases and natural aging. Importantly, this spontaneous process can also modify therapeutic proteins by altering their purity, stability, bioactivity, and antigenicity during drug synthesis and storage. Deamidation occurs non-enzymatically in vivo, but can also take place spontaneously in vitro, hence artificial deamidation during proteomic sample preparation can hamper efforts to identify and quantify endogenous deamidation of complex proteomes. To overcome this, mass spectrometry (MS) can be used to conduct rigorous site-specific characterization of protein deamidation due to the high sensitivity, speed, and specificity offered by this technique. This article reviews recent progress in MS analysis of protein deamidation and discusses the strengths and limitations of common "top-down" and "bottom-up" approaches. Recent advances in sample preparation methods, chromatographic separation, MS technology, and data processing have for the first time enabled the accurate and reliable characterization of protein modifications in complex biological samples, yielding important new data on how deamidation occurs across the entire proteome of human cells and tissues. These technological advances will lead to a better understanding of how deamidation contributes to the pathology of biological aging and major degenerative diseases. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:677-692, 2017.

Proteomic Study of Degenerative Protein Modifications in the Molecular Pathology of Neurodegeneration and Dementia

“…In addition to label-free proteomic methods, isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tag (TMT) protein labeling are widely accepted approaches for quantitative profiling of cell lines and clinical brain tissue samples [24][25][26]. Proteomics has also been used for the accurate identification of protein modifications [26][27][28][29][30][31].…”

“…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 nonenzymatic 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.…”

“…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 modifications including deamidation cause age-related changes in biological functions and play major role in aging.…”

“…Hence, an accurate identification of DMPs and modification 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]. Loss of synapses is one of the most significant contributors to the cognitive impairment manifest in VaD and other neurodegenerative diseases.…”

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

“…The surface exposed amino acid residues, which are solvent accessible, are highly prone towards chemical modifications involving electrophilic and nucleophilic reactions. This is commonly observed for amino acids like lysine, arginine, asparagine, glutamine and cysteine with reactive functional nucleophilic group [3,17]. Furthermore, the asparagine and glutamine residues are highly prone to pH and temperature mediated deamidation [3,18,19].…”