Objective: Recombinant human Erythropoietin (rh-EPO), a 34 kDa glycoprotein, is the most used biopharmaceuticals in the world. In this work, from a candidate national standard developed for rh-EPO a methodology was established to evaluate impurities and to elucidate the structure of peptides in protein digests by UPLC/HDMS E .
Methods:Peptide mapping and intact protein analysis were performed. For peptide mapping specificity and selectivity were increased based on the active ion mobility separation by T-wave device inside the mass spectrometer in positive ion mode with electrospray ionization and UPLC configured with HSS T3 column, flow rate: 800 uL/min, column temperature: 60˚C mobile phase A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile. The batch was digested trypsin after reduction and alkylation.Reduced protein was injected using Massprep Intact protein analysis kit. For intact protein analysis was performed using micro desalting column and 1,5 ug injected. The data was processed using Biopharmalynx software to confirm the protein sequence.
Results:The raw spectrum of rh-EPO tryptic peptides was processed and 99,4 % rh-EPO sequence was verified. The deconvoluted mass spectrum is showed for intact protein analysis and information on heterogeneity was measured. The glycosylation sites were also identified in N-linked N24, N38 and N83 and O-linked S126 which is in agreement with the literature.
Conclusion:It was demonstrated that candidate could be used as national standard for rh-EPO final product and peptide mapping identification. The characterization of rh-EPO peptide maps was showed with high sequence coverage and was successful identified in 80 the batch. The results clearly show the benefits in terms of software and data analysis by mass spectrometry and UPLC to sequence confirmation and post translational modification.
Mining is a high-risk activity due to its dangerous processes. Tin (Sn) is obtained from cassiterite ore and mining activities expose workers to the metal. Chronic exposure to Sn may cause pneumoconiosis, gastrointestinal and hematological effects, among others. This work aimed to assess the exposure of workers to tin in a cassiterite ore processing industry, using the speciation analysis in blood plasma. Twelve subjects donated the blood samples; six were occupationally exposed to Sn. Size exclusion chromatography separated proteins in blood plasma; a graphite furnace atomic absorption spectrometer determined total tin in the plasma and eluted fractions, while SDS-PAGE determined molecular masses of proteins. Tin levels in the workers’ plasma were four times higher than in the reference individuals. After fractionation, the metal only appeared in the total inclusion volume, not being possible to confirm the binding of tin to proteins, which certainly modifies their functions and impair workers’ health. Despite that, the work process needs to change since Sn levels in the workers’ plasma pointed to metal exposure. Further works are necessary to clarify whether the metal is free or bound to small proteins in blood plasma and understand the true impact of tin on workers’ health.
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