ABSTRACT
Deamidation is one of the most common degradation impurities in protein and peptide drugs. The deamidation of glutamine and asparagine in the protein sequence can lead to changes in the chemical and biological properties of the protein. However, the rutine trypsin-based pretreatment process can significantly increase the artificial deamidation impurities during the digestion process, resulting in high determination level. In this study, after optimizing the conditions of Glu-C enzymatic hydrolysis, we obtained the best enzymatic conditions under acidic condition and the artificial deamidation impurities significantly reduced in digestion process, identified the deamidation site (N48). Through the methodological investigation and comparison of the measurement results of different methods, the specificity, reproducibility and accuracy of the method are verified. The method established in this research has laid a solid foundation for the accurate determination of deamidation impurities in cobratide and its similar protein peptide biochemical drugs.
ABSTRACT
Fluorous solid-phase extraction ( FSPE ) is a solid-phase extraction technique based on fluorous affinity between perfluorous compounds. It requires derivatization on analytes with fluorous tags and further specific separation accomplished by perfluorinated solid phase. This technique has extended to various research fields with broad application including organic synthesize, catalysis, chemical and biological analysis. Recently, owing to its good compatibility with mass spectrometry, new analytic techniques relying on FSPE coupled to biological mass spectrometry have received wide attention. This review briefly introduced the principle of FSPE and emphasized on its application for the analysis of biomolecules with mass spectrometry, as well as its outlook of future development.
ABSTRACT
@#Quantitative proteomics is a mass spectrometry-based toolkit used to analyze and quantify entire proteins contained in whole cells, tissues or organisms. It has become an increasingly important element in exploring the mechanism of various biological processes such as discovering novel biomarkers and unknown drug targets. Emerging advances in biological mass spectrometry instrumentation and data acquisition methodologies have provided a state-of-the-art platform for protein quantification, prompting the research of proteomics evolving from the simple qualitative to the accurate quantitative approach. This review aims to introduce the most recent advancements in mass spectrometry instrumentation and methodologies of data acquisition, focusing on their characteristics and applying fields. It also highlights several significant applications of biological mass spectrometry in pharmaceutical research such as quantifitation of drug transporters and metabolizing enzymes, and pharmacokinetic study of therapeutic peptides and proteins.
ABSTRACT
As the complexity of samples and experimental processes, the repeatability of mass spectrometry experiments is still not satisfactory, the results of peptide identification and quantification show high randomicity), the probability of peptide being detected by mass spectrometry in proteome research, especially in quantitative proteomic study, has received much attention. Therefore, a lot of experimental researches have been done, as well as a number of computational prediction methods have been developed. In this article, we summarized the important factors impacting the peptide detectability, investigated the existing prediction methods) and reviewed their applications in experimental study.